Script for Automated One Step Forming Analysis(automotive22-e)

iber Recoating and Proof TestingA Complete Series of Products forCoating Restoration and Reliability TestingAdvantages• Flexible Splice Protection• High Density Splice Packaging • Long Term Reliability• High Volume Production ProcessingPTR-200-ARLModule Configurations• MRC - Manual Fiber Recoater with Auto Injection • XLR - Extended Length Recoater with Auto Injection • ARC - Fully Automated Fiber Recoater • RPT - Rotary Proof Tester / Tension Tester • LPT - Linear Proof Tester• MRL - Combination Manual Recoater & Linear Proof Tester • MRR - Combination Manual Recoater & Rotary Proof Tester • ARL - Combination Automatic Recoater & Linear Proof TesterManual Recoat Mold Automatic Recoat MoldPTR-200-MRLThe rotary optical fiber proof tester / tension testerfeatures Vytran’s rotating mandrel design. To proofor tension test a fiber, the section of fiber to be testedis located between two mandrels. The ends of thefibers are wrapped once around each mandrel andPTR-200-RPTAll Modules offer a choice of control/display interface. The standard interface is a touch screen panel display that provides system status and programming capabilities. Vytran also offers a mini-controller display, which provides system status only. The mini-display is a common option for repetitive production applications where the operator does not need to change process parameters. If process parameters do need to be changed on a system equipped with a mini-controller, Vytran offers a hand-set controller that can be temporarily connected to the machine for re-programming. All modules can also be controlled directly over their RS-232 interface, providing easy integration into existing manufacturing control software.Module Configurations Manual Fiber RecoatingAutomatic Fiber Recoating Automatic Recoat InjectionRotary ProofTestingLinear Proof TestingMRC Module*••XLR Module**••RPT Module •ARC Module*••LPT Module •ARL Module*•••MRL Module*•••MRR Module*•••*Maximum recoat length is two inches (50mm).**Maximum recoat length is four inches (100mm).Module Configuration1400 Campus Drive West • Morganville, NJ 07751 USA • Tel: (732) 972-2880 • Fax: (732) 972-62298 Kew Court Pynes, Hill Office Campus • Rydon Lane • Exeter EX2 5AZ England • Tel: +44 (0) 1392 445777 • Fax: +44 (0) 1392 445009General DimensionsBase Module (except MRR): H: 5.0” (127 mm), W: 10.25” (260 mm), D: 5.0” (127 mm). Base Module (MRR): H: 5.0” (127 mm), W: 10.25” (260 mm), D: 7.0” (178 mm).Control Panel:H: 5.0” (127 mm), W: 8.0” (203 mm), D: 1.0” (25 mm). External Power Supply:H: 2.4” (60 mm), W: 4.1” (105 mm), D:12.5” (320 mm).Electrical RequirementsBase Module: 12.5 VDC, 13 A. (Provided by external powers supply)Control Panel:12.5 VDC, 500 mA. (Provided from base module)External Power Supply:Voltage: 100-120 / 200-240 VAC. Current: 4.5 / 2.2 A. Frequency: 47-63 Hz.Supply RequirementsARC Module:Vacuum for fiber holding blocks (external vacuum pump provided) ARC, ARL Modules:80-120 psi dry compressed air or gas for pneumatic gripper. InterfaceBase Module:RS-232 Serial Communication.RecoatingRecoating ManualAutomatic Modules: MRC, XLR, MRR ARC, ARL Recoat Mold: Quartz.Quartz.Recoat Diameter: 260µm std. for nominal 250µm fiber 280µm std. for nominal 250 µm fiber..Custom sizes from 200µm and up. Custom sizes are available.Maximum Recoat Length: 2” (50 mm) for MRC, MRR Modules.4” (100 mm) for XLR Module.2” (50 mm) for ARC, ARL Modules.Recoat Material: UV Curable Acrylate.DSM 950-200 recommended.UV Curable Acrylate.DSM 950-200 recommended. UV Source:Four 10 W halogen lamps, for MRC. Eight 10 W halogen lamps, for XLR.32 UV LEDsRecoat Injection: Automatic, direct from 1 oz. bottle.Automatic, direct from 1 oz. bottle.Recoat Volume: Programmable in microliters, 5µl typical. Programmable in microliters, 1.5µl typical.Recoat Inject Rate:Programmable, up to 1.8µl/s. Programmable, up to 1.8µl/s. Inject/Lamp-on Delay Time: Programmable, 5s typical. Programmable, 5s typical.Cure Time:Programmable, 17s typical. Programmable, 17s typical.Mold Cleaning Requirement: After every recoat. Start-up and shut-down only.Mold Open/Close Method: Manual. Automatic (pneumatic gripper).Total Cycle Time:60s typical.45s typicalProof TestingProof Testing RotaryLinearModules:RPT, MRRLPT, MRL, ARL Load Application: Rotating Mandrel. Linear Fiber Clamp.Mandrel Diameter: 2” (51 mm). N/AFiber Clamp Length: N/A.1.5” (38 mm).Fiber Spacing:5” (127 mm). 2.9” (74 mm).Min. Fiber Length Req.: 17” (432 mm). 6” (150 mm).Maximum Load: 20 lbs., 9.1 kg.>800 kpsi (5.5 Gpa) for 125µm fiber. 4.5 lbs., 2.1 kg.235 kpsi (1.6 Gpa) for 125 µm fiber.Accuracy:+/- 2%+/- 2%.Ramp Rate:Manual adjust up to 5 lbs/s (2.3 kg/s). Programmable, up to 5 lbs/s (2.3 kg/s).Hold Time:N/A.Programmable.Display Units:lbs, kg, N, kpsi, GPa.lbs, kg, N, kpsi, GPa.PTR-200 Specification。

Selenium Remote Control Information and Installation Procedure Automation Testing: IntroductionSoftware testing using an automatic test program will generally avoid the errors that humans make when they get tired after multiple repetitions. The test program won't skip any tests by mistake. The test program can also record the results of the test accurately. The results can be automatically fed into a database that may provide useful statistics on how well the software development process is going. On the other hand, software that is tested manually will be tested with a randomness that helps find bugs in more varied situations. Since a software program usually won't vary each time it is run, it may not find some bugs that manual testing will. Automated software testing is never a complete substitute for manual testing.Definition:Software testing assisted with software tools that require no operator input, analysis, or evaluation.Benefits:About Selenium:(1) Selenium is an open-source functional testing tool written byThoughtWorks (Read more here:,/selenium). It is the only open-source testing toolthat supports a large variety of browsers and operating systems.(2) It is implemented entirely in JavaScript and runs inside the web browseritself.(3) Selenium Core is a test tool for web applications, its tests run directly in abrowser, just as real users do.About Selenium Core:(1) Selenium Core tests run directly in a browser, just as real users do. Andthey run in Internet Explorer, Mozilla and Firefox on Windows, Linux andMacintosh. No other test tool covers such a wide variety of platforms.(2) Browser Compatibility Testing> Test your application to see if it workscorrectly on different browsers and operating systems. The same scriptcan run on any Selenium platform.(3) System functional testing> Create regression tests to verify applicationfunctionality and user acceptance.About Selenium IDE:(1) Selenium IDE is an integrated development environment for Seleniumtests. It is implemented as a Firefox extension, and allows you to record(that records mouse and keyboard actions as Selenium commands wecan save as test scripts, saving labor and reducing mistakes), edit anddebug tests.(2) Selenium IDE includes the entire Selenium Core, allowing you to easilyand quickly record and play back tests in the actual environment that theywill run.(3) Selenium IDE is not only recording tool: it is a complete IDE. You canchoose to use its recording capability, or you may edit your scripts byhand. With auto complete support and the ability to move commandsaround quickly, Selenium IDE is the ideal environment for creatingSelenium tests no matter what style of tests you prefer.Selenium IDE Features:(1) Easy record and playback.(2) Intelligent field selection will use IDs, names, or XPath as needed.(3) Auto Complete for all common Selenium Commands.(4) Walk through tests.(5) Debug and set breakpoints.(6) Save tests in HTML, Java, C#, Perl, Python, and in Ruby Scripts format.(7) Option to automatically assert the title of every page.Selenium Platform and Browser Compatibility:(1) Supported Platforms:(i) Windows:Known Working:(a) Internet Explorer 6.0 and 7.0(b) Firefox 1.5.0.8 to 2.0(c) Opera 8.5.4 and 9.0.2Should Work:(a) Firefox 0.8 to 2.0(b) Mozilla Suite 1.6+, 1.7+(c) Seamonkey 1.0(d) Opera 8.5+, 9(ii) Mac OS X:Known Working:(a) Firefox 1.5.0.4 to 2.0Should work:(a) Safari 1.3+(b) Firefox 0.8 to 2.0(c) Camino 1.0a1(d) Mozilla Suite 1.6+,1.7+(e) SeaMonkey 1.0Not Yet Supported:(a) OmniWeb(iii) Linux:Known Working:(a) Firefox 1.5.0.8 and 2.0(b) Opera 9.0.2(c) Konqueror 3.5.3Should work:(a) Firefox 0.8 to 2.0(b) Mozilla Suite 1.6+,1.7+(c) Konqueror 3.5+(d) Opera 8.5+, 9Installation Procedure of Selenium-Remote-Control:(1) The Selenium Server is written in Java, and requires the Java Runtime Environment (JRE) version 1.5.0 or higher in order to start. You may already have it installed. Try running this from the command line:java –versionYou should see a brief message telling you what version of Java is installed, like this:java version "1.5.0_07"Java(TM) 2 Runtime Environment, Standard Edition (build 1.5.0_07-b03)Java HotSpot(TM) Client VM (build 1.5.0_07-b03, mixed mode)If you see an error message instead, you may need to install the JRE(/en/download/manual.jsp),Click on …Download‟ button displaying after Windows (Offline Installation) and in stall the Java Runtime Environment.Or you may need to add it to your PATH environment variable if Java Runtime Environment was already installed.For this example, you'll also need to make sure that you have a supported browser installed.Windows:(i) If you're using Windows XP or Windows 2003, you can just use Internet Explorer for this example, or install Mozilla Firefox or Opera.(ii) If you're using Windows 2000, you'll need to install reg.exe in order to use Internet Explorer, but Firefox should work regardless.Add your browser executable to your PATH environment variable. (If you do not explicitly add your browser's installation directory to the PATH, then you must install your browser in its standard location; Firefox's standard location is"c:\Program Files\Mozilla Firefox\firefox.exe"; Internet Explorer's standard location is "c:\Program Files\Internet Explorer\iexplore.exe".)(2) Installing ActivePerl latest build:Go to /Products/ActivePerl/ , download latest active perl build(ActivePerl 5.8.8.819(latest version as on 05/12/2006)) and install it. (3) Installing latest Selenium-Remote-Control build:Go to /selenium-rc/, download latest selenium-remote-control build (0.9.0) zip file and extract all files to a folder (For Ex: Selenium-Remote-Control-0.9.0(latest version as on 05/12/2006)).Place this folder in the drive where ActivePerl was installed.(4) Installing the Selenium Perl Client Driver:Go to /~lukec/Test-WWW-Selenium/, download latest selenium perl client driver (1.10) zip file and extract all files to a folder (For Ex: Test-www-Selenium-1.10)Place this folder in //Perl/bin/Test-WWW-Selenium-1.10 directory where Active Perl was installed.Now, Open command prompt, open //Perl/bin/Test-WWW-Selenium-1.10 (latest version as on 05/12/2006) path in the drive it was installed.(i) You'll need to run Perl on "Makefile.PL" to autogenerate a makefile, run the below command at command promptDrive Letter: \Perl\bin\Test-WWW-Selenium-1.10> perl Makefile.PL(ii) Then run “nmake” command at command prompt.Drive Letter:\Perl\bin\Test-WWW-Selenium-1.10> nmakeNote: Do not specify any filename after nmake. By default it takes Makefile.PL as a file parameter.Small Reference:Makenmake - If you download modules from CPAN, you will often need to installthem with the 'make' utility. NMAKE15.EXE is a self-extracting zip file. When it'sexecuted, it will create 3 files on your disk (nmake.exe, nmake.err, and readme.txt). Move nmake.* to c:\perl\bin, which should be on the path. Then you can run this standard perl command set for installing modules:(iii) then run “nmake test” command at command prompt.Drive Letter:\Perl\bin\Test-WWW-Selenium-1.10> nmake testAfter running this command, displays results with total number of tests, tests passed, tests failed, % of tests failed. You need not worry about these statistics. This is a bug filed under Selenium-Remote-Control. But, still you can go ahead from this step and execute Automated Test Script.(iv) and then run "nmake install" to install it into your Perl distribution.Drive Letter:\Perl\bin\Test-WWW-Selenium-1.10> nmake installNote that this Makefile is incompatible with cygwin's GNU make; on Windows you should use nmake instead. (Note that nmake comes along with ActiveState Perl; it should already be available in your Perl\bin directory.)(4) Using the Selenium Perl Client Driver:Once you've installed the Perl Client Driver in one way or another, you'll need to use its modules. Just as there's more than one way to install the Perl Client Driver, there is also more than one way to use it.First, make sure you've already started the Selenium Server separately in another process. The Selenium Server should remain up and running throughout this process; you shouldn't need to start/stop it each time you use the Client Driver. (Though, of course, if you need to start and stop the server, you certainly can, just by automatically starting it from the command line.)Go to command prompt, open the drive where …Selenium-Remote-Control-0.9.0‟ Run the below sequence of commands at command prompt:Drive Letter: \cd Selenium-Remote-Control-0.9.0Drive Letter:\Selenium-Remote-Control-0.9.0> cd ServerDrive Letter: \Selenium-Remote-Control-0.9.0\Server>java –jar selenium-server. jar –port 8080Note: Please make sure the port number should be same in Selenium automated test script and when starting the selenium server jar file.If you're going to use the driver for automated testing, you may prefer to use Test::WWW::Selenium instead. Test::WWW::Selenium subclassesWWW::Selenium and provides convenient testing functions, suitable for use with Test::More. Test::WWW::Selenium does not require explicit start/stop commands (since these will be handled during test cleanup), and allows you to quickly make any Selenium method a test simply by adding the suffix "_ok" to any Selenium method. Hence, instead of using $sel->click you can use $sel->click_ok to make it a test. In addition, for each Selenium getter (get_title, ...) there are six autogenerated methods (<getter>_is, <getter>_isnt, <getter>_like,<getter>_unlike, <getter>_contains, <getter>_lacks) to check the value of the attribute).How to execute Selenium Perl Scripts:(1) Created a folder …Smoke Tests‟ in \\Perl\bin\ where Active Perl was installed, keep all your smoke test scripts in …Smoke Tests‟ folder.(2) Make sure the port number defined in Perl Test Script and in Selenium Server jar file is same.(3) Start the Selenium-server.jar file from command prompt.For Ex: Drive Letter:\Selenium-Remote-Control-0.9.0\Server\Java Selenium-server.jar –port 8080(4) Open another command prompt and follow the below steps to execute selenium perl script:Change to Smoke Test Scripts directory from root where active perl was installed. Drive Letter:>cd Perl\bin\Smoke TestsDrive Letter:\Perl\bin\Smoke Tests>Execute perl test script from command prompt by typing the following command Drive Letter:\Perl\bin\Smoke Tests>Perl filename.plDefinitions:Selenium RC - Selenium Remote Control.Client Driver - A program that intends to send commands to the browser. Normally the Client Driver is an automated test. The Client Driver is normally not a web browser. Normally a Client Driver relies on a Server to transmit commands to the browser.Client - A Client Driver. The term "Client" by itself is discouraged, because it is easily confused with the browser.Driver - A Client Driver. The term "Driver" by itself is discouraged, because it is easily confused with the Client and the Server considered as a single unit.Server - A program designed to accept HTTP requests from Client Drivers and respond with the results of those commands.Selenium Core - The product that defines the core HTML/JS standard for Selenium. It defines all Selenium Commands that can be performed as well as the communication protocol between the Selenium Server and the Browser. Selenium Command - An browser-level action that is handled by Selenium Core.Server Command - A Selenium Command to be handled by the Selenium Server itself, and is not handled by Selenium Core.Command - A generic term describing either a Selenium Command or the Server Command.Command Request - A request initiated by the Client Driver to the Server, requesting that the Server perform a Command (possibly by passing it to Selenium Core) and return the result.Command Response - The Server's response to the Client Driver's Command Request.HTTP Parameters - Name/value pairs that have been URL encoded in the manner of an HTML form data set. In HTTP GET requests, HTTP Parameters MUST appear in the URL of the request. In HTTP POST requests, HTTP Parameters MUST be encoded in the body of the HTTP request. (Surprisingly, this standard is not defined in any HTTP or URL RFC, but is only defined in the specifications for HTML [1].)Parameters - A short name for HTTP GET Parameters. When used in this way, "Parameters" will always be capitalized.Browser Session - A testing session associated with a web browser. Commands may be run on a particular Browser Session.Session ID - A string identifying a Browser Session.Result Body - The body of the HTTP Command Response.Accessor - A Command that is designed to return data (one or more strings, numbers, booleans) to the Client Driver. ("Accessor Command" is redundant, but the term may be used for clarity.)Accessor Request - A Command Request for an Accessor.Accessor Response - A Command Response from an Accessor Command, whose result body will contain the Accessor's returned data.Action - A Command that is not an Accessor. An Action returns no data. Action Request - A Command Request for an Action.Action Response - A Command Response from an Action.References:(1) For complete reference about selenium automated testing tool/(2) For configuration of perl client with selenium/src/LUKEC/Test-WWW-Selenium-1.10/README (3) For complete selelnium perl scripting commands/selenium-rc/perl/index.html。

SAP for UFT OneRelease SAP continuously but with less risks and costs by accelerating and simplifying your func-tional testing efforts using one intelligent solution with embedded AI-based capabilities.Product HighlightsY our SAP T esting ConundrumWhatever part of the SAP journey you are on, from keeping up-to-date with the latest service packs and updates, creating innovations with the SAP Business T echnology Platform (SAP BTP) through to a total transformation with a S/4HANA Cloud migration automated testing is a key strategy for delivery. As providing your business with the innovation and competi-tive advantage it requires means they need to have confidence in the quality of changes made. T raditional methods can’t give you what you need as using Business employees to con-duct expensive and slow manual testing won’t keep pace or provide you the level of assur-ance you need. Cutting corners by using free/ cheap tools or believing the false hopes and promises of newer tools, is only going to lead to disappointment due to higher than expected maintenance costs.How UFT One Can HelpOpenT ext UFT One is the perfect compan i on for your SAP testing journey as it ac c elerates and simplifies all the related functional testing with one intelligent solution that automates testing for SAP using native AI-based capabilities. UFT One is proven over many successful projects to be the right option for SAP testing teams whatever their distribution or makeup; its specifically designed to work best with non-technical business type testers but it also provides flexibility for the experienced testing professionals through their own specific inter-faces. UFT can efficiently scale testing across distributed infrastructure to best suite however your SAP Application is deployed be it a SAPGUIor SAPGUI for HTML based On Prem ECC or aFiori RISE S/4HANA Private and Public CloudEditions or any of the SAP Cloud Applicationslike SuccessFactors or Concur, etc.If you have a mobile interface, no problem,you can test in parallel on web and mobile,with script once and replay all tests with crossbrowser support. Leverage the broad ecosys-tem of integrations, from version control likeGit, to continuous integration like Jenkins.SAP UFT includes complete native integrationwith SAP Solution Manager and gCTS includ-ing important SAP capabilities like SAPUI5,NetWeaver Business Client, WebDynpro ABAP,CRM UI, SuccessFactors, Hybris, iDocs, RFC& OData plus many other SAP and additionalsupport of 200+ GUI and API, non SAP technol-ogies. This means you can create end-to-endtests against your chosen Business Processregardless of technologies used for true en-terprise grade automated testing.Key BenefitsAccelerating End-to-End T estingof Enterprise-Grade AppsDominate the tide of technologies with a singletool for end-to-end testing and integration ofmultiple technologies, environments, and apps.COMPREHENSIVE TECHNOLOGY STACKAutomated testing of more than 200+ GUI andAPI technologies across mobile, web, desk-top and mainframe, including SAP, Salesforce,Java, Citrix and more.Data SheetData Sheet SAP for UFT OneTRUE END-TO-END TESTINGFROM A SINGLE TOOLCentralize functional and regression testing across all layers of enterprise architectures, all designed to extend tests—from the UI to the API, including web, mobile, composite and packaged apps.EXTENSIVE SAP SUPPORTUFT One offers extensive support for SAP Fiori (SAPUI5 objects and methods), SAPGUI for HTML, WebDynpro ABAP, NetWeaver Business Client for HTML & Desktop, CRM UI and Enterprise Portal. SAP business ap-plications such as Ariba, Concur, Fieldglass, Success Factors and Hybris. Plus, API’s like SOAP, OData, RFC & iDocs.SAP CERTIFIED INTEGRATIONUFT has its own direction integration into SAP Solution Manager Test Suite which is certi-fied by SAP. This is simple to setup and quick to start using meaning you can create, store, edit and execute UFT scripts from Solution Manager, plus it contains the ability to pass test results and bidirectional parameters as part of the integration which ensures that your tests in Solution Manager and UFT are always in sync.AI-BASED TEST AUTHORING AND EXECUTIONUFT One combines AI-based object recogni-tion, object interaction, and natural languagescript creation, to allow tests to be quicklywritten and executed on multiple differentplatforms without requiring any modification,increasing test accuracy, resiliency, and veloc-ity. Not only does UFT AI work for Web basedSAP Applications, like Fiori but also for automa-tion of the the desktop based SAPGUI.REMOTE AI SERVICEInstall the UFT One Remote AI Service on onecentral powerful computer to provide fasterand more efficient AI capabilities to less pow-erful UFT One machines.Boost Productivity withIntelligent AutomationUFT One’s intelligent automation capabilitiesenable teams using UFT One to accelerate thecreation of automation assets and reduce themaintenance effort required to match the paceof application changes.SYNTHETIC DATA CREATIONCreate data intelligently using multiple al-gorithms to reduce the size of a test data setand reduce the time to test, without seriousloss of quality.DATA-DRIVEN ASSETSThe T est Combinations Generator (TCG) cre-ates reusable test data on-the-fly using happy/ error paths and multiple algorithms. DataCapture enables one to pull lists of data val-ues directly from an app into UFT One with afew simple clicks. Customer Data Templateenables for the creation of a custom xml tem-plate that generates parameter types for a test.The Undo/Redo T oggle allows one to quicklyundo or redo multiple steps while configuringdata for a test.Efficiency at Scale—T est MorePer Cycle in Less TimeAchieve test execution at full velocity. Bursttests across distributed functional testing in-frastructures and run tests at scale with fullparallel, cross-browser and cross-device mo-bile testing.CROSS-BROWSER COVERAGEScript once and replay all tests seamlesslyacross the leading browsers and browserversions, including Chrome, Chromium Edge,Firefox, Safari, IE, and Edge. T ests can be re-corded on one browser and the same scriptcan be used with no adaptions to test multiplebrowsers and configurations. If desired, thesame test run can cover all the different brows-ers by having each test iteration executed on adifferent browser, with one report covering thetest flow status on all desired configurations.CLOUD-BASED DEPLOYMENTT o expand your footprint, simply deploy UFTOne in the cloud on your provisioned Citrix,AWS, and Azure virtual environments.SHIFT-LEFT TESTING WITH UFT DEVELOPER*Increase productivity with a shift-left test auto-mation tool created for Developers using theIDE, language and testing framework of choice__________*U FT Developer is available for use with UFT Oneat no additional cost.to create tests at the same time the application is being developed. Also, support Developer / QA collaboration by converting UFT One ob-ject repositories to UFT Developer application models, or by converting UFT Developer appli-cation models to UFT One object repositories. MOBILE TESTING ON LOCAL DEVICESThe UFT Mobile Add-in for Local Devices in-tegrates UFT One and mobile devices con-nected directly to the UFT One host machine. In just a few steps, UFT One users can start de-signing and running mobile app and web tests on local mobile devices without purchasing an additional license. This type of tight integration allows UFT One users to execute omnichan-nel content strategies using the same script for desktop and mobile web, support mobile testing in companies that do not yet have a lab management solution in place, or run mo-bile tests without requiring a new tool for mo-bile devices.SummaryUFT One is part of the industry leading UFT family of integrated Functional T esting solu-tions which enables customers to test earlier and faster by combining a breadth of tech-nology support with AI-driven capabilities to deliver the speed and resiliency required to achieve automation at scale that is tightly in-tegrated with an organization’s current Dev-Ops toolchain.Learn more atOpenT ext Solutions for SAP Software Modernization | OpenT ext/opentext。

ConSAT user manualVersion 1.0 – March 2014Alfonso E. RomeroDepartment of Computer Science, Centre for Systems and Synthetic BiologyRoyal Holloway, University of London – Egham Hill, Egham, TW20 0EXTable of ContentsConSAT user manual (1)1. Overview of ConSAT (2)2. The Software (2)2.1 Requirements (2)2.2 Installation (2)2.3 Features (3)3. A quick introduction to ConSAT (3)4. How to use ConSAT (4)4.1 The ConSAT modes: manual and automatic (4)4.2 Running GFam (5)4.3 Understanding the representation of an architecture (5)4.4 Associating functional labels with ConSAT (5)5. Output files (5)6. The ConSAT algorithm (6)7. The GFams dataset (6)8. Version History of this software (6)1. Overview of ConSATConSAT stands for “Consensus Signature Architecture Tool”. Using ConSAT on a set of protein sequences you can get their corresponding protein families. The protein families, for our purpose, are consensus domain architectures.Proteins within the same family are assumed to share many properties among them as they are assumed to descend from a common ancestor –that is, they are evolutionarily related. Thus, protein families can be of a good help in the study of large protein sets, moving us from the study of single sequences to the study of the set of families (the number of families found will be lower than the set of individual sequences).The way ConSAT builds the architectures is by combining two sources of data:(1)domain assignments from InterPro, and (2) domain assignments from GFams (see section 7. The GFams dataset). The combination is done in a way that no overlapping domains will be allowed, and maximising the sequence coverage. See section 6. The ConSAT algorithm for more details. ConSAT is written in Python and has a few requirements to run properly(see section 2.1 Requirements). You can check, as well about its installation (section 2.2 Installation) or its set of features (2.3 Features). To learn how to run it, there is a quick introduction in section 3. A quick introduction to ConSAT, and a more detailed manual in 4. How to use ConSAT. Details on the output files are given in 5. Output files. The final section of this manual, 8. Version History of this software, shows the historical changes to the ConSAT software for the different versions of it. ConSAThasbeenmainlydevelopedbyAlfonsoE.Romero<****************>with the contribution of Tamás Nepusz, Rajkumar Sasidharan, and Alberto Paccanaro. This project has been funded by the Biotechnology and Biological Sciences Research Council (BBSRC).2. The Software2.1 RequirementsIn order to run properly, ConSAT requires the following programs to be installed in your machine:•Python 2.7.•NCBI Blast +.•HMMer version 3.0 (or higher).•Scipy: is not needed by recommended for efficiency purposes.2.2 InstallationFirst of all, download the sources of the stable release from /software/consat or check the unstable release at https:///alfonsoeromero/ConSAT/ (they eventually might be the same).To install this software the same procedure for installing any Python module(See /2/install/#inst-new-standard for more details) can be followed:•Unpack the sources•Go to the newly created folder•Run the command python setup.py install•Sometimes the previous step requires superuser (root) permissions. For Linux systems like Ubuntu this can be done by pre-pending sudo to the previous command, or entering a superuser terminal (executing su).After installation, the command consat should be available at any directory of your system. Please check very carefully that this is the case by opening a terminal, running consat and check that no errors other than the lack of parameters are produced.2.3 Features2.3.1Software architectureConSAT is a set of Python basic scripts, each one prepared to perform a single task, and a set of master scripts to run the whole pipeline. The basic scripts take a set of input text files and produce another set of output files. The master scripts take a configuration file with the routes of all the input files needed and the value of the parameters and run a whole ConSAT computation. The master script is relying on modula, a very powerful Python coded by Tamás Nepusz which avoids doing all the calculations (in case something goes wrong) by storing several intermediate files. Thus, any ConSAT calculation requires a work directory (where the intermediate files will be created) and an output directory (where the output files will be written). After any typical ConSAT execution you will only be intersted in looking at the output directory.2.3.2Hardware requirementsTo process an average set of sequences (20-30,000 protein sequences) you will need nothing more than a regular machine. Any nowadays computer with a few gigabytes of hard drive, and 4-8 gigabytes of RAM will do the trick. We will try to use several processors if they are available in the parts of the computation which can be run in parallel.If you want to run ConSAT in a larger set of sequences (>100,000 sequences) you will probably be able to do this, but the RAM consumption would be increased a bit. Depending on your dataset you may need 16 or 32 gigabytes of RAM to make ConSAT run properly. Some tasks are done in memory as this is a compromise between efficiency and space usage (and we prefer the latter). However, if you want to run ConSAT in a single organism it is very uncommon that you need more than 8 GB of RAM.2.3.3LicenseConSAT is released under the GNU General Public License (GPL) v3 license. You can check this license online here: /licenses/gpl.html. A copy of the license has been provided in the ConSAT sources.3. A quick introduction to ConSATconsat is driven by a master configuration file. You can create an empty configuration consat file by typing:$ consat initThis will produce a file consat.cfg in the same directory where you run consat. In order to properly run the ConSAT pipeline, open the configuration file and modify the fields according to your needs. There is a proper description of each field in that file, explaining what it is for and what values you can put it there. After this, you can re-run ConSAT with your own configuration file(assuming this is config.cfg and still is located in the same directory) by doing: $ consat -c config.cfgThis will run ConSAT and will show in the standard output the progress of the calculations. Depending on the length of your set of protein sequences this could take from a few minutes to a few hours.4. How to use ConSATThis is the “long” version of the mini-manual presented in section 3. A quick introduction to ConSAT. Here we will go into deeper detail and will explain further options beyond the basic ones.4.1 The ConSAT modes: manual and automaticConSAT can be used in two different modes: the manual and the automatic one. In the manual mode, the user will have to proceed as stated in section 3. A quick introduction to ConSAT: a configuration file will have to be created, the parameters will have to be filled in, and finally, ConSAT will be run with that configuration file. Assuming success in the execution, the output files will be located in the output folder.There is even an easier way to run ConSAT, and this is the automatic mode. While in the manual mode the user could specify the values of the parameters, in the automatic mode there is no such possibility (except for a few options). However, in the automatic mode ConSAT will download almost every needed data file, and will free the user from this tedious task.To run ConSAT in the automatic mode,you will use the script automated_consat.For instance, having a FASTA file of proteins named proteins.fasta, an InterPro annotation of these proteins in proteins.interpro and a GFams release in gfams_1.0.hmm, we can run the following command:$ automated_consat -s proteins.fasta -i proteins.interpro -m gfams_1.0.hmm consat resultThe work files, the downloaded data and the output of ConSAT will be written to the result directory (last parameter). With the parameter “consat” we are noting that the automated script will run ConSAT. There is, as well, the possibility of an “automated GFam mode” (see next section).All the needed files will be downloaded in their more recent versions: the Gene Ontology file, the translation table between InterPro terms and GO terms(interpro2go),the descriptions for the domains from other databases (Pfam, Panther, etc.), and the hierarchical relations between the different InterPro domains. In the user-specified result directory, a data directory will be created containing the download files,and two directories(work and output)will contain the intermediate and the final produced files, respectively. A configuration file will be created, as well, in the user-specified result directory. Thus, if the same command is run in a different moment within a different result directory, it is not guaranteed that the results are 100% the same (because new data files, different from the first ones, could have been downloaded into your data directory, therefore changing your final results).If you want to reproduce your results from a previously run experiment in automatic mode you should do the following:•Get your data directory and your consat.cfg file from the result folder and move them into a different place (or even, different computer).•Run ConSAT with that configuration file, making sure that the routes in that file correspond to the routes of the data folder, wherever you have placed it.4.2 Running GFamHaving installed ConSAT, it is possible to run Gfam (/software/gfam/) almost as in the original way. After the installation, the executable gfam should be available. This will allow to run GFam on your set of sequences with the following differences:•The efficiency of the process should have improved with respect to the original version.•Some bugs have been changed from the original release (eventually they should be corrected in the GFam software as well).•The treatment of domain insertions is completely new, and they are explicitly represented as insertions in the corresponding output files.•HMMs are learnt from the clusters found.In order to do so,the software packages clustal-omega and HMMer are needed.• A method for associating functions transferred from proteins in a GOA file having the same architecture has been developed. Therefore, new options in the configuration file have been added.• A method for combining the functional labels obtained by over representation of GO terms obtained from InterPro-assigned domains to the architecture plus those obtained from transferred from a GOA file is available (you would have an extra file in the output folder). If you want, you can run GFam in an automatic mode, using the same automated_consat script with the following format:$ automated_consat -s proteins.fasta -i proteins.interpro gfam resultNote that, in this case, no models should have to be provided (GFam does not need the Gfams HMM models), and the mode will be now gfam (the parameter before the last). Everything which we pointed out for the “automatic” mode of ConSAT holds too for this GFam mode.4.3 Understanding the representation of an architectureIn all the output files regarding protein architectures we give a textual representation of the architecture. An architecture is either NO_ASSIGNMENT (no architecture was found for that protein sequence) or a protein architecture expression.Protein architecture expressions are composed of three things (being the second and the third optional):1.Protein domain names (e.g. IPR00001, PTH024420, GFAM00010).2.Semicolons (“;”)3.Curly braces (“{“ and “}”).Semicolons separate protein domains found one next to the other.Thus,the architecture IPR00001;PTH024420can be understood as the domain IPR00001followed by the domain PTH024420. Semicolons mark domain juxtaposition, and should be used between any two domains appearing in the protein domain architecture.Curly braces mark that the containing domain (or list of domains) was found inside the preceding domain. If we find an architecture like this: IPR00001;PTH024420{IPR00007} this means that the domain IPR00001 is followed by the domain PTH024420, inside which a hit of the domain IPR0007 was found.For computer scientists, this is the BNF grammar for protein domains we are using in ConSAT:<protein_architecture>::= “NO_ASSIGNMENT” | <domain_expression><domain_expression> ::= <domain> [“;” <domain_expression> ]<domain> ::= <domain_name> [ “{” <domain_expression> “}”]<domain_name> ::= any domain name from the different domain Dbs4.4 Associating functional labels with ConSATWe provide Gene Ontology terms for the protein architectures found. These terms come from two different methods:•Over represented terms from InterPro domains:once the architecture has been found, the set of GO terms associated to the InterPro terms in the architecture is retrieved (using the InterPro2GO table). Once we get this set, an over-representation analysis is carried out over it, and only significant terms are kept (the significance level, by default is 0.05). The over representation analysis is carried out against the InterPro2GO table (this defines de prior distribution for any GO term).•Transferred terms from a GOA file: if we provide a GOA file with functions for some of our proteins, we can use this to assign GO terms to our sequences. The algorithm is the following:For each architecture, we retrieve the set of proteins matching itFor each protein in that architecture, we retrieve its set of assigned GO termsFor each GO term, we carry out an over representation analysis with respectto the GOA file (that defines the background distribution for each GO term)If the test is passed at a certain alpha, the term is assigned to thearchitectureAgain, we use alpha equal to 0.05.We provided, as well, a method combining both set of assigned GO terms. The combined p-value for each GO term is obtained by using Fisher's method(you can check /wiki/Fisher's_method for details). Note that, when a GO term is associated by only one of the methods it will get that same p-value as the combined one.5. Output filesWe provide here a brief description of the most important output files produced by a normalConSAT execution.File domain_architecture_details.txt :In this file, a detail of the architecture assignment for each individual protein sequence is given. The file is in a very easy format, with a few lines for each protein sequence, like this example where two proteins are presented (one with some assignments in its architecture, and another without any architecture assigned). Note that both the original domain and the one assigned by InterPro are given:N1VAX8Primary assignment source: HMMPfamNumber of data sources used: 1Data sources: HMMPfamCoverage: 0.655Coverage w/o novel domains: 0.65515- 257: PTHR24220 (HMMPfam, stage: 1)17- 255: SSF52540 (HMMPfam, stage: 1) (InterPro ID: IPR027417)P-loop containing nucleoside triphosphate hydrolase34- 179: PF00005 (HMMPfam, stage: 1) (InterPro ID: IPR003439 --> IPR027417) P-loop containing nucleoside triphosphate hydrolaseN1VAX9Primary assignment source: NoneNumber of data sources used: 0Data sources:Coverage: 0.000Coverage w/o novel domains: 0.000File domain_architecture s.t ab :Tab separated file with the following fields for each protein:•Protein id•Starting position covered by the architecture•Ending position covered by the architecture•Length covered by the architecture•Architecture specifying the position of each domain (shown with an interval in parentheses).•Description of the architecture formed by the concatenation of the individual domain descriptions (if any).File overrep_by_arch.txt :For each architecture, it shows the functional prediction (GO terms) together with the p-value, which is associated to each architecture via the overrepresentation of GO terms associated by InterPro to each domain.A line with the architecture is shown, followed by the functional predictions for that architecture, indented (if any). Architectures are separated among each other with a double carriage return.For this kind of files, the prediction is shown at the predefined significance level (0.05 by default), but if the file ends in “_unfiltered”, all predictions are shown regardless of their p-value.File transfer_by_arch.txt :Same kind of file that the overrep_by_arch.txt. Functional labels are obtained now by transferring experimental GOA terms from proteins with the same architecture and then running overrepresentation on the set of transferred labels. The format is the same as in the mentioned file. File combined _prediction_by_arch.txt :It gives the predictions given in the files transfer_by_arch and overrep_by_arch combined using Fisher's method. The format is the same as in those files.File weight_file_per_arch.txt :It is a tab separated file with two columns. In the first one we have architectures, and in the second we have a vector of assigned terms (identified by their integer id) and weights, separated by space. For example, this could be a line:IPR007541;IPR000772{G3DSA:2.80.10.50} 3330:6.32311 3:4.48The mentioned architecture has two terms assigned, the 3330 and the 3, weighting 6.32311 and 4.48, respectively. The mapping between term identifiers and term strings is given by the lexicon file.File text/lexicon :Maps the words associated to architectures and their identifiers. For each line it shows a triplet: <word> <word_id> <doc_freq>Where <word> is the word itself, <word_id> is a positive number, unique for each word, and <doc_freq> is the number of documents (sequences) containing this word.6. The ConSAT algorithmWe give here the pseudocode of the ConSAT algorithmFOR each protein p:architecture = {}assignment_length = {}sorted_p = sort_by_length(assignments(p))FOR each InterPro Assignment a in sorted_p:IF not overlaps(a, assignment_length[a.source]):assignment_length[a.source].add(a)primary_source = arg_max(assignment_length)architecture.add_all(assignment_length[primary_source])FOR each stage in Stages:sources = get_sources(stage)s = filter_by_source(assignments(p), sources)s = sort_by_length(s)FOR each InterPro assignment a in s – architecture:IF not overlaps(a, architecture):architecture.add(a)r = find_unassigned_regions(p, architecture)r = r – low_complexity_regions(p)FOR each region in r:hits = hmm_scan(GFams, r)hits = sort_hits_by_evalue(hits)FOR each hit in hits:IF not overlaps(hit, architecture):architecture.add(hit)return architecture7. The GFams datasetThe GFams dataset is a set of putative domains found in UniProt. We say they are putative because they have been obtained by purely computational methods, although we believe they could be protein domains from a computational point of view. The GFams dataset is obtained by the following procedure:1.GFam is applied on a certain SwissProt release.2.The clusters found (see GFam paper) are chosen as domains.3. A HMM is learnt in each cluster to predict future occurrences of this domain.The GFams database has its own release number and we plan to rebuild them from time to time, but not so frequently as the ConSAT database. Given that the rate of growth of SwissProt is much less than the one of the whole UniProt, we think that we do not need to relearn SwissProt with every UniProt release.The HMMs for the GFams are needed for running ConSAT and can be downloaded from the same website of the ConSAT web server: /consat.8. Version History of this software1.0 (March 2014):-first version of ConSAT.。

Rat TNF-alpha Sandwich ELISA Kit DatasheetPlease read it entirely before use Catalogue Number:KE20001Size: 96TSensitivity:7.7 pg/mLRange: 15.6-1000 pg/mLUsage:For the quantitative detection of rat TNF-alpha concentrations in cell culture supernatant.This product is for research use only and not for use in human or animal therapeutic or diagnostic.1/8Table of content page1. Background32. Principle33. Required Materials44. Kit Components and Storage45. Safety Notes46. Sample Collection and Storage47. Regent Preparation58. Assay Procedure Summary69. Validation Data79.1 Standard curve79.2 Precision79.3 Recovery89.4 Sensitivity89.5 Linearity810. References82/81. BackgroundTNF, as also known as TNF-alpha, or cachectin, is a multifunctional proinflammatory cytokine that belongs to the tumor necrosis factor (TNF) superfamily. It is expressed as a 26 kDa membrane bound protein and is then cleaved by TNF-alpha converting enzyme (TACE) to release the soluble 17 kDa monomer, which forms homotrimers in circulation. It is produced chiefly by activated macrophages, although it can be produced by many other cell types such as CD4+ lymphocytes, NK cells, neutrophils, mast cells, eosinophils, and neurons. It can bind to, and thus functions through its receptors TNFRSF1A/TNFR1 andTNFRSF1B/TNFBR. This cytokine is involved in the regulation of a wide spectrum of biological processes including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation. rat and human TNF-alpha share 79% amino acid sequence identity. Unlike human TNF-alpha, the rat form is glycosylated. In rat deficiency of this gene is associated with defects in response to bacterial infection, with defects in forming organized follicular dendritic cell networks and germinal centers, and with a lack of primary B cell follicles.2. PrincipleSandwich ELISA structure (Detection antibodylabeled with biotin)A capture antibody is pre-coated onto the bottom of wellswhich binds to analyte of interest. A detection antibodylabeled with biotin also binds to the analyte. Streptavidin-HRPbinds to the biotin. TMB acts as the HRP substrate and thesolution color will change from colorless to blue. A stopsolution containing sulfuric acid turns solution yellow. Thecolor intensity is proportional to the quantity of bound proteinwhich is measurable at 450 nm with the correction wavelengthset at 630 nm.3. Required Materials3.1 A microplate reader capable of measuring absorbance at 450 nm with the correction wavelength set at 630 nm.3.2 Calibrated, adjustable precision pipettes and disposable plastic tips. A manifold multi-channel pipette is recommended for large assays.3.3 Plate washer: automated or manual.3.4 Absorbent paper towels.3.5 Glass or plastic tubes to prepare standard and sample dilutions.3.6 Beakers and graduated cylinders.3.7 Log-log or semi-log graph paper or computer and software for ELISA data analysis. A four-parameter logistic (4-PL) curve-fit is recommended.3/84. Kit Components and StorageMicroplateMicroplate - antibody coated 96-well microplate (8 well × 12 strips) 1 plate Unopened Kit:Unopened Kit: Store at 2-8°C for 6 months or -20°C for 12 months.Opened Kit:Opened Kit: All reagents stored at 2-8°C for 7 days.Please use a new standardfor each assay.Protein standardProtein standard - 1000 pg/bottle; lyophilized 2 bottles Detection antibody, biotinylated (100×)Detection antibody, biotinylated (100×) - 120 μL/vial*1 vial Streptavidin-horseradish peroxidase (HRP) (100×)Streptavidin-horseradish peroxidase (HRP) (100×) - 120 μL/vial* 1 vial Sample Diluent PT 1-ef Sample Diluent PT 1-ef - 30 mL/bottle 1 bottle Detection DiluentDetection Diluent - 30 mL/bottle 1 bottle Wash Buffer Concentrate (20×)Wash Buffer Concentrate (20×) - 30 mL/bottle 1 bottle Tetramethylbenzidine Substrate (TMB)Tetramethylbenzidine Substrate (TMB) - 12 mL/bottle 1 bottle Stop Solution Stop Solution - 12 mL/bottle 1 bottlePlate Cover Seals4 pieces * Centrifugation immediately before use5. Safety Notes5.1 Avoid any skin and eye contact with Stop Solution and TMB. In case of contact, wash thoroughly with water.5.2 Do not use the kit after the expiration date.5.3 Do not mix or substitute reagents or materials from other kit lots or other sources.5.4 Be sure to wear protective equipment such as gloves, masks and goggles during the experiment.5.5 When using an automated plate washer, adding a 30 second soak period following the addition of Wash Buffer to improve assay precision6. Sample Collection and Storage6.1 Cell Culture Supernatant: Remove particulates by centrifugation for 5 minutes at 500xg and assay immediately or aliquot and store samples at ≤ -20℃. Avoid repeated freeze-thaw cycles.4/87. Regent Preparation7.1 Wash Buffer (1X):7.1 Wash Buffer (1X): If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Add 30 mL of Wash Buffer Concentrate(20X) to 570 mL deionized or distilled water to prepare 1X Wash Buffer.7.2 7.2 Detection Antibody Detection Antibody (1X): (1X): Dilute 100X Detection Antibody 1:100 using Detection Diluent prior to assay. Suggested 1:100dilution: 10 μL 100X Detection Antibody + 990 μL Detection Diluent (Centrifuge the 100 X Detection Antibody solution for a few seconds prior to use).7.3 7.3 Streptavidin-HRP Streptavidin-HRP (1X) (1X): : Dilute 100X Streptavidin-HRP 1:100 using Detection Diluent prior to assay. Suggested 1:100dilution: 10 μL 100X Streptavidin-HRP + 990 μL Detection Diluent (Centrifuge the 100X Streptavidin-HRP solution for a few seconds prior to use).7.4 Sample Dilution:7.4 Sample Dilution: Different samples should be diluted with corresponding Sample Diluent, samples may require further dilution if the readout values are higher than the highest standard OD reading. Variations in sample collection, processing and storage may affect the results of the measurement.Recommended Dilution for different sample types: 1:2 or 1:4 is recommended for cell culture supernatant.7.5 Standard Serial Dilution:Add 1 mL Sample Diluent PT 1-ef in protein standard.5/88. Assay Procedure SummaryBring all reagents to room temperature before use (Detection antibody and Bring all reagents to room temperature before use (Detection antibody and Streptavidin-HRP Streptavidin-HRP can be used immediately). To avoid cross-contamination, change pipette tips between additions of each standard level,between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.8.1 Take out the required number of microplate strips and return excess strips to the foil pouch containing the drying reagent pack and reseal; store at 4°C immediately. Microplate strips should be used in one week.8.2 Preset the layout of the microplate, including control group,standard group and sample group, add 100 μL of each standard and sample to the appropriate wells.(Make sure sample addition is uninterrupted and completed within 5 to 10 minutes, It is recommended to assay all standards, controls, and samples in duplicate).8.3 Seal plate with cover seal, pressing it firmly onto top of microwells. Incubate the plate for 2 hours at 37°C.8.4 Wash1) Gently remove the cover seal. Discard the liquid from wells by aspirating or decanting. Remove any residual solution by tapping the plate a few times on fresh paper towels.2) Wash 4 times with 1X Wash Buffer, using at least 350-400 μL per well. Following the last wash, firmly tap plates on fresh towels 10 times to remove residual Wash Buffer. Avoid getting any towel fibers in the wells or wells drying out completely.8.5 Add 100 μL of 1X Detection Antibody solution (refer to Reagent Preparation7.2) to each well. Seal plate with cover seal and incubate for 1 hour at 37°C.8.6 Repeat wash step in 8.4.8.7 Add 100 μL of 1X Streptavidin-HRP solution (refer to Reagent Preparation7.3) to each well. Seal plate with cover seal and incubate the plate for 40 minutes at 37°C .8.8 Repeat wash step in 8.4.8.9 Signal development: Add 100 μL of TMB substrate solution to each well, protected from light. Incubate for 15 to 20 minutes. Substrate Solution should remain colorless until added to the plate.8.10 Quenching color development: Add 100 μL of Stop Solution to each well in the same order as addition of the TMB substrate. Mix by tapping the side of the plate gently. NB: Avoid skin and eye contact with the Stop solution.8.11 Read results: Immediately after adding Stop solution read the absorbance on a microplate reader at a wavelength of 450nm. If possible, perform a double wavelength readout (450 nm and 630 nm).8.12 Data analysis: Calculate the average of the duplicate readings (OD value) for each standard and sample, and subtract the average of the zero standard absorbance. Construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis, use four-parameter logistic curve- fit (4-PL) analysis to do this. If the samples have been diluted, the OD readout from the standard curve must be multiplied by the dilution factor used. 1Standard and Samples 100 µL 120 min 4 times Cover Wells incubate at 37°C 2Diluent Antibody Solution 100 µL 60 min 4 times Cover Wells incubate at 37°C 3Diluent HRP Solution 100 µL 40 min 4 times Cover Wells incubate at 37°C 4TMB Substrate 100 µL 15-20 min Do not wash Incubate in the dark at 37°C 5Stop Solution100 µL0 minDo not wash-6Read plate at 450 nm and 630 nm immediately after adding Stop solution. DO NOT exceed 5 minutes.6/8(pg/mL)O.D Average Corrected00.0830.0890.086-15.60.1610.1790.1700.08431.250.2360.2470.2420.15662.50.4120.4050.4090.3231250.6330.6510.6420.556250 1.0331.0391.0360.950500 1.5651.6791.622 1.5361000 2.2552.3562.306 2.220Intra-assay PrecisionSample n Mean (pg/mL)SD CV% 120288.817.9 6.2 220598.940.2 6.7 3201,088.179.87.3Inter-assay PrecisionSample n Mean (pg/mL)SD CV% 124238.216.97.1 224374.034.29.2 324716.937.2 5.29. Validation Data9.1 Standard curveThese standard curves are provided for demonstration only. A standard curve should be generated for each set of samples assayed.9.2 PrecisionIntra-assay PrecisionIntra-assay Precision (Precision within an assay) Three samples of known concentration were tested 20 times on one plate to assess intra-assay precision.Inter-assay PrecisionInter-assay Precision (Precision between assays) Three samples of known concentration were tested in 24 separate assays to assess inter-assay precision.7/89.3 RecoveryThe recovery of rat TNF-alpha spiked to three different levels throughout the range of the assay in cell culture supernatant was evaluated.Cell culture supernatant 1:29392-94 1:47571-779.4 SensitivityThe minimum detectable dose of rat TNF-alpha is 7.7 pg/mL. This was determined by adding two standard deviations to the concentration corresponding to the mean O.D. of 20 zero standard replicates.9.5 LinearityTo assess the linearity of the assay, three samples were spiked with high concentrations of rat TNF-alpha in cell culture supernatant and diluted with Sample DiluentSample Diluent to produce samples with values within the dynamic range of the assay.1:2Average% of Expected105 Range (%)102-1071:4Average% of Expected99 Range (%)94-1031:8Average% of Expected96 Range (%)95-961:16Average% of Expected90 Range (%)89-9210. References1. Agbanoma G. et al. (2012) J Immunol. 188: 1307-17.2. Kriegler M. et al. (1988) Cell. 53: 45-53.3. Theiss AL. et al. (2005) J Biol Chem. 280: 36099-109.4. Swardfager W. et al. (2010) Biol Psychiatry. 68:930-41.5. Locksley RM.et al. (2001) Cell. 104(4):487-501.6. provided by RefSeq, Jun 2013.8/8。

外文翻译原文：Injection MoldingMany different processes are used to transform plastic granules, powders, and liquids into product. The plastic material is in moldable form, and is adaptable to various forming methods. In most cases thermosetting materials require other methods of forming. This is recognized by the fact that thermoplastics are usually heated to a soft state and then reshaped before cooling. Theromosets, on the other hand have not yet been polymerized before processing, and the chemical reaction takes place during the process, usually through heat, a catalyst, or pressure. It is important to remember this concept while studying the plastics manufacturing processes and polymers used.Injection molding is by far the most widely used process of forming thermoplastic materials. It is also one of the oldest. Currently injection molding accounts for 30% of all plastics resin consumption. Since raw material can be converted by a single procedure, injection molding is suitable for mass production of plastics articles and automated one-step production of complex geometries. In most cases, finishing is not necessary. Typical products include toys, automotive parts, household articles, and consumer electronics goods.Since injection molding has a number of interdependent variables, it is a process of considerable complexity. The success of the injection molding operation is dependent not only in the proper setup of the machine hydraulics, barrel temperature variations, and changes in material viscosity. Increasing shot-to-shot repeatability of machine variables helps produce parts with tighter tolerance, lowers the level of rejects, and increases product quality (i.e., appearance and serviceability).The principal objective of any molding operation is the manufacture of products: to a specific quality level, in the shortest time, and using repeatable and fully automatic cycle. Molders strive to reduce or eliminate rejected parts in molding production.For injection molding of high precision optical parts, or parts with a high added value such as appliance cases, the payoff of reduced rejects is high.A typical injection molding cycle or sequence consists of five phases;1. Injection or mold filling2. Packing or compression3. Holding4. Cooling5. Part ejectionPlastic granules are fed into the hopper and through an in the injection cylinder where they are carried forward by the rotating screw. The rotation of the screw forces the granules under high pressure against the heated walls of the cylinder causing them to melt. As the pressure building up, the rotating screw is forced backward until enough plastic has accumulated to make the shot. The injection ram (or screw) forces molten plastic from the barrel, through the nozzle, sprue and runner system, and finally into the mold cavities. During injection, the mold cavity is filled volumetrically. When the plastic contacts the cold mold surfaces, it solidifies (freezes) rapidly to produce the skin layer. Since the core remains in the molten state, plastic follows through the core to complete mold filling. Typically, the cavity is filled to 95%~98% during injection. Then the molding process is switched over to the packing phase.Even as the cavity is filled, the molten plastic begins to cool. Since the cooling plastic contracts or shrinks, it gives rise to defects such as sink marks, voids, and dimensional instabilities. To compensate for shrinkage, addition plastic is forced into the cavity. Once the cavity is packed, pressure applied to the melt prevents molten plastic inside the cavity from back flowing out through the gate. The pressure must be applied until the gate solidifies. The process can be divided into two steps (packing and holding) or may be encompassed in one step(holding or second stage). During packing, melt forced into the cavity by the packing pressure compensates for shrinkage. With holding, the pressure merely prevents back flow of the polymer malt.After the holding stage is completed, the cooling phase starts. During, the part is held in the mold for specified period. The duration of the cooling phase depends primarily on the material properties and the part thickness. Typically, the part temperature must cool below the material’s ejection temperature. While cooling the part, the machine plasticates melt for the next cycle.The polymer is subjected to shearing action as well as the condition of the energy from the heater bands. Once the short is made, plastication ceases. This should occur immediately before the end of the cooling phase. Then the mold opens and the part is ejected.When polymers are fabricated into useful articles they are referred to as plastics, rubbers, and fibers. Some polymers, for example, cotton and wool, occur naturally, but the great majority of commercial products are synthetic in origin. A list of the names of the better known materials would include Bakelite, Dacron, Nylon, Celanese, Orlon, and Styron.Previous to 1930 the use of synthetic polymers was not widespread. However, they should not be classified as new materials for many of them were known in the latter half of the nineteenth century. The failure to develop them during this period was due, in part, to a lack of understanding of their properties, in particular, the problem of the structure of polymers was the subject of much fruitless controversy.Two events of the twentieth century catapulted polymers into a position of worldwide importance. The first of these was the successful commercial production of the plastic now known as Bakelite. Its industrial usefulness was demonstrated in1912 and in the next succeeding years. Today Bakelite is high on the list of important synthetic products. Before 1912 materials made from cellulose were available, but their manufacture never provided the incentive for new work in the polymer field such as occurred after the advent of Bakelite. The second event was concerned with fundamental studies of the nature polymers by Staudinger in Europe and by Carohers, who worked with the Du Pont company in Delaware. A greater part of the studies were made during the 1920’s. Staudinger’s work was primarily fundamental. Carother’s achievements led to the development of our present huge plastics industry by causing an awakening of interest in polymer chemistry, an interest which is still strongly apparent today.The Nature of ThermodynamicsThermodynamics is one of the most important areas of engineering science used to explain how most things work, why some things do not the way that they were intended, and why others things just cannot possibly work at all. It is a key part of the science engineers use to design automotive engines, heat pumps, rocket motors, power stations, gas turbines, air conditioners, super-conducting transmission lines, solar heating systems, etc.Thermodynamics centers about the notions of energy, the idea that energy is conserved is the first low of thermodynamics. It is starting point for the science of thermodynamics is entropy; entropy provides a means for determining if a process is possible.This idea is the basis for the second low of thermodynamics. It also provides the basis for an engineering analysis in which one calculates the maximum amount of useful that can be obtained from a given energy source, or the minimum amount of power input required to do a certain task.A clear understanding of the ideas of entropy is essential for one who needs to use thermodynamics in engineering analysis. Scientists are interested in using thermodynamics to predict and relate the properties of matter; engineers are interested in using this data, together with the basic ideas of energy conservation and entropy production, to analyze the behavior of complex technological systems.There is an example of the sort of system of interest to engineers, a large central power stations. In this particular plant the energy source is petroleum in one of several forms, or sometimes natural gas, and the plant is to convert as much of this energy as possible to electric energy and to send this energy down the transmission line.Simply expressed, the plant does this by boiling water and using the steam to turn a turbine which turns an electric generator.The simplest such power plants are able to convert only about 25 percent of the fuel energy to electric energy. But this particular plant converts approximately 40 percent;it has been ingeniously designed through careful application of the basic principles of thermodynamics to the hundreds of components in the system.The design engineers who made these calculations used data on the properties of steam developed by physical chemists who in turn used experimental measurements in concert with thermodynamics theory to develop the property data.Plants presently being studied could convert as much as 55 percent of the fuel energy to electric energy, if they indeed perform as predicted by thermodynamics analysis.The rule that the spontaneous flow of heat is always from hotter to cooler objects is a new physical idea. There is noting in the energy conservation principle or in any other law of nature that specifies for us the direction of heat flow. If energy were to flow spontaneously from a block of ice to a surrounding volume of water, this could occur in complete accord with energy conservation. But such a process never happens. This idea is the substance of the second law of thermodynamics.Clear, a refrigerator, which is a physical system used in kitchen refrigerators, freezers, and air-conditioning units must obey not only the first law (energy conservation) but the second law as well.To see why the second law is not violated by a refrigerator, we must be careful in our statement of law. The second law of thermodynamics says, in effect, that heat never flows spontaneously from a cooler to a hotter object.Or, alternatively, heat can flow from a cooler to a hotter object only as a result of work done by an external agency. We now see the distinction between an everyday spontaneous process, such as the flow of heat from the inside to the outside of a refrigerator.In the water-ice system, the exchange of energy takes place spontaneously and the flow of heat always proceeds from the water to the ice. The water gives up energy and becomes cooler while the ice receives energy and melts.In a refrigerator, on the other hand, the exchange of energy is not spontaneous. Work provided by an external agency is necessary to reverse the natural flow of heat and cool the interior at the expense of further heating the warmer surroundings.译文：塑料注射成型许多不同的加工过程习惯于把塑料颗粒、粉末和液体转化成最终产品。

One Step 的使用方法在Autoform 中，onestep 功能可以快速的查看制件的成型性，从而加快设计和规划进程。

下面图1所示为onestep 的功能选项卡。

一步法的选择是在geometry generator 下将数型进行处理后，在进入input generator 时进行选择的。

图2中的Simulation type 选项中下边的选择项就是一步法。

当点击此项后，会弹出图3所示对话框。

点击OK 即可进入图1所示界面。

图1图2 图3Geometry 选项卡1.类型(Type)一步法中有五种模拟方式可选。

任何一种方式下都要对OS boundary 选项进行设置，此项用来给出拉延结束后制件的边界线，而非拉延前的坯料线。

此处统一说明，后边重复处不再做解释。

A 无补充无压边圈如图4所示为此种方式。

由于没有压边圈也没有补充，因此binder 选项和OS punch opening 选项是不可选的。

此种方式是最简单也最粗糙的一种算法。

B 无补充有压边圈图5所示为此中方式。

此方式激活binder 选项，若在geometry generator 中就生成压边圈，则此选项会自动将其添加。

由于一步法多用来粗算，压边圈多采用自动生成，因此此处会自动添加。

计算相对上一种方式也多了压边圈闭合过程。

图4图5C 无补充有分模线如图6中所示，此种方式需要输入产品最终边界线和分模线。

其计算过程中没有补充。

D 有补充无压边圈图7中为此方式。

与上一种方式相比较此方式没有压边圈，因此没有压边过程。

E 有补充有压边圈图6图7图8此方式下需要给出分模线和拉延后的坯料线。

以上五种方式从前向后逐步增加复杂程度。

BLANK选项卡如图9所示，此选项卡下需输入料厚以及材料种类。

也可以增加称线以及板料的冲孔等。

Process选项卡图10和图11给出了此选项卡的设置内容。

限制条件的类型共有自由、锁死、约束、压边压强和压边力五种可选项。

当没有压边圈时，后两项是不可选的。

Models: RPR3006W RadiPower ®Product ManualRF Power MetersPage 2 of 19RadiPower® product manualThis product manual pertains to the RadiPower®.Models: RPR3006W - RPR3008W - By Raditeq 16/08/21All trademarks used in this manual are the property of their respective ownersSupplier InformationRaditeq B.V.Vijzelmolenlaan 33447 GX, WoerdenThe NetherlandsTel.: +31 (0)348 200 100Internet: Email: *****************Read this manual carefully before operating the product and make sure all the safety instructions are strictly followed.For your convenience, a Quick Start Guide has been added to this product. This Quick Start Guide contains the basic start-up steps and the safety warnings.Please keep the Quick Start Guide (and this regular manual) close at hand when you operate your new Raditeq product(s).Please contact your local reseller if you have any questions.Table of contentsWARNINGS & PRECAUTIONS 4 Introduction 5 The RadiPower® Series 6 Product characteristics 6 Functional description 7 The RadiPower® 3000 Series 8 Model RPR3006W 8 RadiPower® Installation 9 Stand-alone Configuration 9 Software Configuration 10 How to configure the RadiPower® in RadiMation® 10 How to connect the RadiPower® to the RadiCentre® 11 How to check whether the RadiPower® is properly connected to RadiMation® 11 Adding coupler values in RadiMation®12 Theory of operation in burst mode 13 Prefix & Communication example: 13 Remarks about the general commands 14 Stand-alone command set 14 Burst Mode 15 Specifications RPR3006W16 Warranty Conditions18Page 3 of 19WARNINGS & PRECAUTIONSRead the contents of this product manual carefully and become familiar with the safety markings, the product instructions and the handling of the system. Please refer to the applicable product manual(s) for further information regarding the operation and control of the product(s).Only Raditeq qualified maintenance personnel is allowed to perform maintenance and/or repair service on the equipment.This product® contains materials that can be recycled and reused to minimize material waste. At the ‘end-of-life’, specialized companies can dismantle the discarded system to collect the reusable and recyclable materials. If your product is at its ‘end-of-life’, please return it to your local reseller or to Raditeq for recycling.For cleaning, use a clean, dry cloth (or a damp cloth where needed) and wipe the surface of equipment.This product contains no hazardous substances as described in the RoHS Directive (2015/863/EU).This product contains embedded software, which is field upgradeable.For more information contact your local reseller or go to Page 4 of 19IntroductionThis manual contains information about the RadiPower® RPR3006W RF power meters.An accurate power meter is indispensable to perform reliable EMC measurements. The RadiPower® is a RF power meter especially designed for power measurements during EMC tests. The RadiPower® is an affordable, accurate and fast power meter. It provides accurate measurements over a wide frequency range, which enables effective measurements in accordance with the latest EMC standards.Please read this manual carefully and make sure to pay special attention to the chapters regarding your new product(s).RadiCentre® SystemThe RadiCentre® is a modular EMC/RF test system that serves as the interface between user and computer for all the RadiCentre® plug-in cards and modules.RadiMation® SoftwareRadiMation ® is the EMC software package from Raditeq used for remote controland automated testing of the RadiCentre® plug-in cards and modules and is sold separately. RadiField® Electric Field GeneratorThe patented RadiField® Triple A is no less than a revolution in EMC immunity testing.A complete paradigm shift involves a combination of high-level integration anda field combining technique, making several discrete components like combiner,coupler, power meters and cabling superfluous. This product is sold separately.RadiMationShielded USB cable Model: USB A male to USB mini B5 male.To connect the RF power sensor to a USB connector.The RadiPower® 3000 SeriesMMCX Trigger cable For measurements on multiple ports.RadiPower® RF power sensor Model: RPR3006W.Supporting documentation is supplied on a USB stick containing:•The (digital) User Manual and Quick Start Guide.•The installation of RadiMation® Free software and drivers.• Optional - The calibration certificate for the power meter.The RadiPower® RF power sensor is optimized for EMC measurements, where a high dynamic range, together with fast measurements, are required even at low power levels. Where most power sensors require long measurement times at low RF levels, the RadiPower® RF power sensor is able to perform accurate power measurements, with a high measurement speed, at power levels close to the noise floor, without the need for zero adjustment!Fast - EMC immunity measurements are time consuming. This is mainly dependent on the number of frequency points, the dwell time and the speed of the power meter. As the first two parameters are generally prescribed by standards, the only one that can be optimized is the speed of the power meter. The unprecedented detector technology of Raditeq’s power meters makes extremely fast and accurate power measurements a reality, even at low power levels.Accurate - Accuracy is another concern in addition to speed, when performing EMC measurements. The RadiPower® allows for high precision EMC measurements with a large dynamic range. Because the RadiPower® has a high accuracy over the complete band, it is suitable for measurements in accordance to automotive, military, telecom and EMC basic standards such as the IEC61000-4-3/6 standards.Simultaneous power measurements on multiple ports - The RadiPower®RPR3006W has two trigger ports (MMCX) which can be used to synchronizemultiple power meters. These power meters connected through a daisy chaintrigger each other to make time synchronized measurements. Due to this functionmeasurements of WLAN and/or MIMO devices according to the EN 300 328 andEN 301 893 standards.All necessary parameters can be set and calculated by RadiMation® Free (or ahigher version) making the setup and measurements effortless.Low measurement uncertainties - Impedance mismatches contribute to themeasurement uncertainty. The RadiPower® has a very low Standing Wave Ratio(SWR) and as a result, measurement uncertainties are low compared to othercontributions in the EMC measurement setups.Robust housingThe RadiPower® RF power sensor is mounted in a rugged metal housing to ensure a long life and excellent RF shielding. The power sensor is equipped with an N-type precision RF input connector and a mini USB-B connector for communication with a computer.Easy to use - With the USB interface the RadiPower® is easy to use. In addition, the RadiPower® can be controlled by both the RadiMation® integral EMC measurement software and any other EMC measurement packages, because all the software commands needed to control the unit are available. For ‘stand-alone use’ of the RadiPower®, RadiMation® Free measurement software is delivered with the power meters. The RadiPower® 3000 SeriesProduct characteristicsFunctional descriptionThe RadiPower® uses a high speed power detector to measure the RF signal, independent of the crest factor of the input signal waveform. The detected signal is sampled, at high speed, by a high speed ADC and the samples are processed by a powerful DSP. The sophisticated software enables unique functions, such as envelope tracing and burst logging. The table below shows which models support the different measurement modes.0 : CW1 : Peak √√2 : Envelope tracing√3 : Burst√CW modeThe RadiPower® performs RMS power measurements of CW-signals. In RMS mode the RadiPower® samples the signal at high speed. The RMS value of the power is calculated over the number of samples defined by the filter setting and can be read by a simple command. Due to the high sampling speed the number of readings is high, even at large filter settings. This mode is supported for all the RadiPower® models.Peak modePeak mode keeps track of the maximum power that is measured during a specific time interval. In PEAK mode, the “power?”-command will return the highest value that was measured since the last “power?”-command. After each cycle of the command the previous peak value is cleared,Burst modeIn burst mode, the RadiPower can measure a burst of RF power. It is even possible to measure a burst on multiple powermeters at the sime time (using the MMCX trigger ports). The power readings of each powermeter can be combined to a total power by RadiMation according to the EN 300 328 and EN 301 893 standards..In this mode the sample speed can be set to either 1 MS/s or 5 MS/s. The RadiPower can store a total of 100.000 bursts and is independent of the observation time. The stored data of a single burst consists of the RMS power and the start/stop-time of the burst. The start and stop time are defined by how long the power was higher than the threshold value. The threshold value can be set in RadiMation.The RMS power of a burst is calculated according to the following formula:Where m is the number of RMS power samples within the start and stop time. When measuring on a MIMO device with more than one port. It is possible to use multiple power meters. The burst values of each power meter can be combined to a single RF power using RadiMaiton. This can also be done manual by using the following formula:i is the number of power metersIt is also possible to add corrections for the coupler, beamforming gain (Y) and the assembly gain (G) of the DUT. RadiMation can compensate for these values.Connect the RadiPower® sensor to a Windows computer with a USB port for ‘stand-alone’ use. Use the supplied USB cable to connect the sensor to your computer. Windows will prompt that new hardware has been found. The USB-driver for the RadiPower® is Windows certified and will be loaded automatically from the Windows update. If the drivers are not loaded automatically, these can be installed manually from the supplied USB-key. Follow the normal instructions from Windows to install the drivers manually.Once the drivers are loaded successfully, the RadiPower® will be shown in the device list.RadiPower® InstallationStand-alone Configuration Connect the RadiPower® sensor to a Windows computer with a USB 2.0 compatible port. Use the supplied USB cable to connect the RadiPower® sensor. The hardware installation for the RadiPower® sensor is now complete.Software ConfigurationIn order to control the RadiPower® from a computer, the RadiMation® EMC software package can be used. RadiMation® from Raditeq is sold separately. A free version of RadiMation® is available on: https://www. /automated-emc-software/radimation-free/.If the RadiPower® is operated manually, this chapter can be skipped.The RadiPower® device driver is part of the Power Meter Device Driver familyHow to configure the RadiPower® in RadiMation®1. Start the latest version of RadiMation®; https:///radimation-download/2. Select the button ‘Device’ at the top menu bar, followed by clicking ‘Configure’;3. In the configuration screen select ‘Device Drivers’ and Select ‘Power meter’ or any of the otherdrivers as driver type;4. Click the ‘Add’ button to open the selection of available drivers in RadiMation®;5. Enter ‘RadiPower’ in the search bar which will show all available RadiPower® drivers;6. Select the correct driver, double click it (Optional, rename it) and press ‘OK’.2354How to check whether the RadiPower® is properly connected to RadiMation®1. Select ‘Devices’ in the top menu bar2. Open ‘Device Drivers’ and select device driver type: ‘Power Meter’3. Double click the recently configured RadiPower® or click ‘Edit’.4. Finally select the ‘Check’ button on the right side of the opened screen.5. When correctly configured, RadiMation® will notify you that the device is correctly installed.23Adding coupler values in RadiMation®In a MIMO test setup, power measurements are often performed using a coupler on the antenna port. Coupling factors can be compensated by RadiMation® using a correction file. If no couplers or additional attenuators are used in the setup, there is no need to create and apply these correction files.To create a correction file, select “File” – “New” – “Correction” from the menu bar.Press “Colums/units” to create to columns for Frequency and Attenuation.To create a constant correction versus frequency:• Press “Add Row” and enter the lowest frequency with the corresponding attenuation.• Press “Add Row” again and enter the highest frequency with the corresponding attenuation.If calibration data of the coupler is available, this data can be used to create an accurate correction for the coupler.After all data has been entered, select “File” – “Save Correction As” to save the data.Next, the correction data of the coupler has to be added to the correct power meter. Select “Configuration” and “Configuration” again. In the Configuration window select the “Device drivers” tab and select the power meter for which the correction has to be added. Press “Edit”.Press the “file open”- button in de Correction Files area to select the correction file. Perform this procedure for each power meter for which a coupler correction has to be added. RadiMation® will now automatically calculate the power values, including the coupler values.Stand-alone command setCommunication with the RadiPower® sensor is possible using a virtual COM-port (VCP)For more information see chapter ‘stand alone configuration’.NOTE: All commands can be found in the RadiCentre® Manual under the programming manual.Specifications RPR3006W & RPR3008W1. In burst mode only 1 and 5 MS/s can be set and used.• All specifications are measured after 10 minutes warm-up time and 0dBm unless specified otherwise.• T ypical specifications indicate that the measured values a re met on at least 80% of the points.• Three years warranty will be granted only after you register the product at . Without registration, a 1 year warranty period applies.For more information about the current and new Raditeq products at:T:+31348200100M:*****************W: Warranty ConditionsRaditeq B.V. offers a standard warranty term of three (3) years on their products, calculated from the shipping date, under the condition that the product is registered on . For registration of the product, the customer should provide the product model, serial number and the responsible reseller (if applicable). If the product is not registered, a limited warranty term of one (1) year will be applicable.Return Material Authorization (RMA) & Warranty repairIf a defect occurs to our product within the warranty term, a Return Material Authorization (RMA) ‘Warranty Repair’ request can be issued using the RMA link at /support. Upon receipt of the request, an RMA number will be provided. Please do not send the product without this RMA number! The defective product should be shipped to our service department at the following address:Raditeq B.V. – Service DepartmentVijzelmolenlaan 33447GX WOERDENThe NetherlandsThere will be no charge for repair services (materials or labour) within the (extended) warranty term.These warranty terms are not applicable to:• Normal wear and tear• Fibre optic cables• Products that have been improperly used• Products that have been used outside their specified range• Products that have been improperly installed and/or maintained• Products that have been modified without approval of Raditeq• Calibration and/or re-calibration of the productRepair services on products that are not covered by the Raditeq warranty will be charged to the customer.Repairs outside warrantyIf a defect is not covered under warranty, an RMA fixed-repair can be ordered on the RMA link: /support If a re-calibration is needed after repair, this calibration should be ordered separately. The calibration will be performed at the ISO17025 accredited calibration laboratories of DARE!! Calibrations, based on the applicable service code / prices. Warranty after repairFor repairs outside the original warranty period, a limited warranty of six months is applicable on the performed repair. Shipping conditions are the same as with repairs that are covered within the original warranty period.ShippingThe customer will need to arrange shipping and cover for the costs (like e.g. transportation costs, duties, taxes) for sending the defect product the service department of Raditeq in The Netherlands. Raditeq will arrange the courier and cover for the costs for the return shipment after repair.EU Declaration of Conformity WeRaditeq B.V.ofVijzelmolenlaan 3NL-3447GX WoerdenThe Netherlandsdeclare under our sole responsibility that theProduct: RadiPower® Seriesmodels: RPR3006Ware in accordance with the European directives:EMC Directive 2014/30/EULow Voltage Directive 2015/35/EURoHS Directive: 2015/863/EUper the provisions of the applicable requirements of the following harmonized standards: Emission: EN 61326-1:2013, Class A1Electrical equipment for measurement, control and laboratory use.Immunity:EN 61326-1:2013, Industrial level, performance criteria AElectrical equipment for measurement, control and laboratory use.Safety:EN 61010-1:2010, Safety requirements for electrical equipmentfor measurement, control, and laboratory useThe technical construction files are maintained at the adress specified above.Date of issue:16/08/21Place of issue:Woerden, The NetherlandsAuthorized by:P.W.J. DijkstraTitle of authority: DirectorRaditeq B.V. | Vijzelmolenlaan 3 | 3447GX Woerden | The Netherlands | T:+31 348 200 100。

Product ManualISP-09-001Contents3 Introduction5ComponentsULAG ravityTRAP™8microtissuesG enerating3D8requiredmaterialsAdditional9 Preparation9 Pre-wetting10 Microtissueseedingmaturation11 Sedimentation/SpheroidMedium exchange in the GravityTRAP™ ULA Plate 12 Analysis and assays in the GravityTRAP™ ULA Plate 13 Annex 1: Microtissue harvest from GravityTRAP™ ULA Plates 14 Annex 2: Troubleshooting guide 16 Annex 3: Step-by-step protocol for HCT116 & HEY microtissues 17Version 2.0, July, 2015451-0009-01-B23IntroductionThe G ravityTRAP™ U ltra-L ow A ttachment(ULA) plate 1 represents a simple, flexible,and automation-compatible platform for thegeneration, long-term cultivation, observationand testing of scaffold-free 3D microtissuespheroids in 96-well format. Each plateconsists of a special non-adhesively coated96-well, sterile-packaged GravityTRAP™ ULA Plate and lid.InSphero recommends G ravityTRAP™ ULA plates for the generation of spheroids using immortalized or modified cell lines that are known to readily form microtissues, or as a starting point for investigating whether or not a cell line can form self-aggregating, scaffold-free spheroids. InSphero recommends our patented GravityPLUS™ Hanging Drop System (ISP-06-001, ISP-06-010) if generating spheroids in more complex 3D cell culture scenarios, such as when using primary cells, cell lines that are sensitive to self-assembly, or when generating co-culture microtissues (e.g., tumor/stroma). In such cases, the GravityPLUS™ Hanging Drop System provides the greatest opportunity for success.1 The GravityTRAP™ ULA Plate and GravityPLUS™ Plate and related technology are protected by several granted and pending patentsworld-wide.4Spheroid formation in the GravityTRAP™ ULA Plate begins with initial seedingAdvantages of the GravityTRAP™ ULA Plate:1. Convenient scaffold-free formation of spheroids via cellular self-assembly inultra-low attachment (ULA-treated) plates2. SureXchange™ tapered ledge and culture chamber facilitates easy mediumexchange and prevents microtissue loss during long-term spheroid growth and analysis3. 1 mm diameter flat bottom observation chamber enables simple spheroidobservation, and greater working field-to-field distance reduces well-to-well imaging cross-talk compared to standard 96-well plates4. 3D-optimized protocols available for analysis in GravityTRAP™ ULA Plate5GravityTRAP™ ULA Plate ComponentsThe complete G ravityTRAP™ ULA Plate assembly consists of the following components:1. Bottom GravityTRAP™ ULA Plate (96-well) (A)2. Lid (B)Figure 2:ULA PlateThe GravityTRAP™ ULA PlateThe G ravityTRAP™ (T issue R e-aggregation and A ssay P late) ULA Plate is a special non-adhesively coated 96-well microtiter plate. It is designed to accomodate production of 3D microtissues (spheroids) for convenient long- term cultivation and analysis. G ravityTRAP™ tapered wells feature a SureXchange™ ledge to prevent inadvertent microtissue aspiration and disruption during medium exchange and compound dosing (Fig. 3). Microtissues are posi-tioned in a 1.0 mm observation chamber at the bottom of each well, which enables automated imaging processes (Fig. 4). Biochemical assays as well as optical analytical methods such as inverse bright field and fluorescence microscopy can be performed.Microtissue production with G ravityTRAP™ ULA Plates is very simple, and recommended for cell lines that are known to readily form spheroids in ULA conditions, or as a first step in characterizing the spheroid-forming capabilities of a particular cell type of interest. A cell suspension is delivered to the bottom plate using a multi-channel pipette or a robotic liquid handler. Following brief centrifugation to concentrate cells near the bottom of the tapered chamber, microtissues begin forming by gravity-enforced self-assembly. Spheroid maturation typically occurs within 2-5 days of seeding depending on the cell type and culture conditions (Figs. 1 & 4).67Figure 3: Bottom plate of theGravityTRAP™ Tissue Re-aggregation and Assay Plate.Figure 4: HCT-116 colon carcinoma microtissue cultured inGravityTRAP™ ULA Plate. Pic ture acquisition with a ZeissAxiovert25 microscope, 5× objective.Generating 3D microtissuesG enerating 3D microtissues in the G ravityTRAP™ ULA Plate is a straightforward process, but one that must be optimized for each cell type. Cell type, growth medium, and intended downstream applications will impact the starting density and desired culture volume. Optimization is recommended for each cell type and application. In addition to the process overview in this chapter, Annex 3 illustrates the formation of spheroids using HCT-116 (human colon carcinoma) and HEY (human ovarian carcinoma) cell lines as an example for optimizing your own protocol.Additional materials required1. Mammalian cells (primary or cell line) of interest2. 3D InSight™ Tumor Microtissue Media Kit (InSphero, cat. no. CS-17-001-01) -includes 3D InSight™ Tumor Re-aggregation Medium (CS-07-111-02) and 3D InSight™ Tumor Maintenance Medium (CS-07-112-01)3. Inverted microscope with a 5x/10x objective4. Cell counter, e.g. Neubauer chamber5. 8-or12-channel pipette(e.g.Viaflo10.0-300.0μl,Integra Biosciences,2 incubator 37°C8Preparation1. Prior to seeding, pre-warm the 3D InSight™ Tumor Re-aggregation Medium(CS-07-111-02).2. Wipe the GravityTRAP™ ULA Plate bag with 70% EtOH before opening.3. Carefully open the bag under sterile working conditions and take out theGravity T RAP™ ULA Plate assembly.Pre-wettingIMPORTANT – Pre-wetting the wells of the GravityTRAP™ ULA Plate according Array to the procedure below is required prior to seeding microtissues to prevent inclusion of air bubbles.IMPORTANT - Perform all of the following steps under sterile conditions.1. Apply 40 µl of 3D InSight™ Tumor Re-aggregation Medium (CS-07-111-02)to each well by placing the tip far into the wells. It is recommended to use a multichannel pipette (8- or 12-channel).2. Remove pre-wetting solution by placing the tip at the ledge of the uppercavity of the well (Fig. 5). Aspirate until medium is completely removed fromeach well. A negligible amount of medium (<5-7 µl) may remain in the bottomof the chamber.910Microtissue seeding1. Trypsinize cells expanded in cell-culture flasks according to your standard pro-tocol.2. Count the cells to determine cells per ml of medium.3. Prepare a cell suspension for seeding, using a final volume per well of 70 µl.Recommended cell concentration: For long-term growth profiling start with low cell numbers (250–500 cells per well). If non-proliferating cells or rap-id production of larger microtissues is required then start with 2500–25000 cells/70 µl. See Annex 3 for a detailed example using HCT-116 (colon carcinoma) and HEY (ovarian carcinoma) cell lines.IMPORTANT – For homogeneity of forming microtissues, it is essential to assure a homgeneous distribution of the cell suspension by gently pipettingup and down prior to seeding into the GravityTRAP™ ULA Plate.Figure 5: Medium removal fromGravityTRAP™ ULA well.4. Gently (≤10 µl/sec) add 70 µl of the cell suspension to the GravityTRAP™ ULA Plate by placing the pipette tips far into the wells (avoid touching the well bottom).Cell sedimentation and spheroid maturationFollowing seeding, it is recommended (but optional) to briefly centrifuge the plate to remove any air bubbles, and to force cells to the bottom of the well in order to promote aggregation and spheroid formation.1. Place the lid on the G ravityTRAP™ ULA Plate and spin in a microtiter-platecentrifuge for 2 minutes at 250 RCF.2. Following centrifugation, remove the plate and incubate the plate in ahumidified CO 2 incubator at 37°C on a level surface for 2-5 days, checking daily to observe microtissue maturation and exchanging medium as necessary. See next section for details on medium exchange.Medium exchange in the GravityTRAP™ ULA PlateThe special GravityTRAP™ ULA Plate design allows routine medium exchange for longer-term cultivation without the risk of microtissue loss. The SureXchange™ ledge at the inside wall of the well serves as an anchoring point for the pipette tip.1. Place the pipette tip at the ledge of the well (Fig. 6, left).2. Remove the medium at low pipetting speed (<30 µl/sec) by aspirating an ex-cess of volume. A minimal volume of ~5-7 µl medium will remain in the well. 3. Add 70 µl of fresh medium by placing the pipette tip at the ledge (Fig. 6 right).Use a dispensing rate <50 µl/sec.Figure 6: Medium ex-c hange in the Gravity-TRAP™ ULA Plate. Left:Medium removal with thepipette tip plac ed at theledge of the well. Right:Medium addition.IMPORTANT – When using automated liquid handling devices, an off-centeralignment of the vertical pipette tip will achieve the same affect. Contact**************************************.Analysis and assays in GravityTRAP™ ULA PlateThe GravityTRAP™ ULA Plate format is compatible with a broad variety of biochem-ical methods and allows for spectrometrical measurements with a multiwell plate reader or for visual inspection of microtissues by an inverted microscope (similar to analysis of standard 2D cultures):Biochemical AssaysSeveral validated biochemical assay protocols for microtissues in the GravityTRAP™ ULA Plate are available. Please see Technical Protocols in the support section of InSphero’s website: /support.HistologyMicrotissues are amenable to analysis by histology. Please request our Technical Protocol TP006.Fluorescent/luminescent multiwell plate reader compatibilityGrowth changes and profiles in tumor microtissues expressing GFP/RFP can easily be analysed using fluorescent plate readers, as the signal intensity is stronger than with monolayer cultured cells.Automated imagingas the SCREEN Cell3Whole mount fluorescent stainingFixed or live cell imaging with fluorescent antibodies. Please request our Technical Protocol TP008.Annex 1:Microtissue harvest from GravityTRAP™ ULA Plates1. Place the pipette tip (1000 µl pipette tip) in a vertical position to cover the cen-ter of the lower cavity. The tip orifice is positioned slightly above the microtis-sue on the well bottom (the larger 1 ml tip size prevents the disruption of the microtissue by being squeezed inadvertently as the tip diameter exceeds the size of the well bottom; Fig. 7A).2. Collect the microtissues by aspirating 50 µl of the medium. Avoid aspiration ofair bubbles, which may provoke the loss of microtissues in the tip.3. Alternatively use a 100-200 µl pipette tip and aspirate 50 µl by placing thehead of the tip close to the bottom of the well (Fig. 7B). Do not touch the mi-crotissues as they will be squeezed and stick to the pipette tip (Fig. 7C).4. Transfer the microtissue into another vessel or plate format.Figure 7: Microtissue harvest from GravityTRAP TM ULA Plate.Annex 2: Trouble-shooting guideAnnex 3: HCT-116 and HEY microtissue formation – A step-by-step protocolThe following protocol describes the production of HCT-116 (human colon carcino-ma) and HEY (human ovarian carcinoma cell line) microtissues using GravityTRAP™ ULA Plates.Materials• Cryopreserved HCT-116 (ATCC CCL-247™) and HEY cells, ideally 1×106 cells per vial• 3D InSight™ Tumor Microtissue Media Kit (InSphero, cat. no. CS-17-001-01) - includes 3D InSight™ Tumor Re-aggregation Medium (CS-07-111-02) and 3D InSight™ Tumor Maintenance Medium (CS-07-112-01)• Cell-culture flasks T75 (Greiner, cat no. 658175)• GravityTRAP™ ULA Plate (InSphero, cat no. ISP-09-001)• Sterile phosphate buffered saline (PBS)• Neubauer chamber• Water bath (37°C)• Serological pipettes, 5 and 10 ml• Plate centrifuge• Level 1 biosafety cabinetincubator 37°C• Humidified CO2• Inverted phase-contrast microscope• 15 ml Falcon tube• Sterile multichannel medium reservoir• Multichannel pipette (e.g. Integra Viaflo 8-channel pipette, InSphero, cat. no.IS-001-01; if not available, a single-channel pipette can be used as well) HCT-116/HEY expansionPerform all following steps under aseptic conditions for each cell line undera laminar flow bench. Procedures are the same for both HCT-116 and HEYcells except where otherwise indicated.1. Ensure that all cell-culture material is in place and labeled.2. Prepare T75 flask by adding 5 ml of 3D InSight™ Tumor Maintenance Medium(CS-07-112-01).3. Fast thaw one vial of each cell line at 37°C in the water bath.remaining 1 ml medium in the pipette and add to the 15 ml tube.6. Centrifuge the cells at 200 RCF for 2 min, remove the supernatant andresuspend the cell pellet with 5 ml of 3D InSight™ Tumor MaintenanceMedium (CS-07-012-01).7. Transfer resuspended cells into the pre-filled T75 flask.8. Place the cell-culture flask into the incubator.9. After 24 hours of incubation replace the medium and check under the micro- scope if cells have adhered on the plastic surface.10. After reaching 70-80% confluence (approx. 48 hours) cells are ready for microtissue production.HCT-116/HEY microtissue formationPrepare the GravityTRAP™ ULA Plate as described on page 9.1. Take the T75 flasks with the HCT-116 or HEY cells out of the incubator.2. Remove medium with the aspiration pipette.3. Add 10 ml PBS.4. Remove PBS.5. Add 1 ml Trypsin EDTA (1×).6. Incubate at 37°C for 5 minutes.7. Ensure that the cells are completely detached.8. Stop trypsinization by adding 9 ml of 3D InSight™ Tumor Maintenance Medium(CS-07-112-01).9. Transfer the cell suspension into a 15 ml Falcon tube.10. Centrifuge for 2 minutes at 200 RCF.11. Control pellet and aspirate supernatant.12. Re-suspend cells in 3D InSight™ Tumor Re-aggregation Medium (CS-07-111-02).13. Determine cell number with the Neubauer chamber (or alternative method).14. Adjust cell number with 3D InSight™ Tumor Re-aggregation Medium(CS-07-111-02) to a density of 1.43×105 cells/ml corresponding to a highest-density stock of 10,000 cells/70 µl) for each cell line; prepare similar stocks of each cell line to create 2500, 500, and 100 cells/70 µl stocks; prepare a sufficient amount of each cell suspension to account for the number of desired replicates at each dilution.15. Pre-wet wells of GravityTRAP TM ULA Plate as instructed on page 9.16. Transfer cell suspension to a medium reservoir. Obtain a homogeneousTM ULA Plate. Seed 70 µl of cell suspension/well using a multi-ravity-TRAP TM ULA Plate for212 minutes at 250 RCF.18. Incubate plates on a level surface in a humidified CO 2 incubator for 3 days andobserve for spheroid formation using brighfield microscopy.19. Typical HCT-116 and HEY spheroids formed with varying cell densities usingthis method are depicted below (Fig. 8). Spheroid size (Fig. 9) and ATP content (Fig. 10) were assessed at day 3 using the SCREEN Cell 3iMager and Promega CellTiter-Glo ® assays, respectively.Figure 8GravityTRAP™ ULA Plate at various c ell densitites, demonstrating cshaped microtissue formation.22Figure 9: Average size (day3) of HCT-116 and HEYtumor spheroids as assessedby volume using theCell3iMager. Standard de-viation of 6 replicates per celldensity shown for eac h c ellline.Figure 10: Average ATPcontent of HCT-116 and HEYtumor spheroids as assessedby Promega CellTiter-Glo®assay. Standard deviation of6 replic ates per c ell densityshown for each cell line.Annex 4: GravityTRAP TM ULA Plate limited use label license This License Agreement (the “License Agreement”) is a legal agreement between the end user (“End User”) and InSphero AG or its subsidiaries (“InSphero”) to use the GravityTRAP™ ULA Plate covered by patents owned or controlled by InSphero which are provided to you.1. Warranties The End User hereby irrevocably warrants to keep and use the GravityTRAP™ ULA Plate in accordance with the restrictions and limitations contained in this License Agreement.2. Proprietary Rights The GravityTRAP™ ULA Plate may be covered by one or more pending or granted patent applications. By entering into this Licence Agreement, End User acknowledges that the GravityTRAP™ ULA Plate is so covered.3. Excluded Fields No permission is granted hereunder for the use of the GravityTRAP™ ULA Plate:1. for selling cell-based products generated using the GravityTRAP™ ULA Plate to thirdparties, or2. in veterinary applications, in diagnostics, in vivo use in humans and/or uses related tofood products (together the “Excluded Fields”).4. Use by the End User Subject to Clause 3 above End User will use the GravityTRAP™ ULA Plate solely for in vitro research in-house for the discovery and development of compounds outside the Excluded Fields by End User.End User will not sell, transfer, disclose or otherwise provide access to the GravityTRAP™ ULA Plate to any third party or entity. End User will not sell or transfer cell-based products generated using the GravityTRAP™ ULA Plate to any third party or entity.5. Disclaimer of Warranties InSphero herby disclaims all representations, conditions and warranties of any kind, express or implied, including, without limitation, any warranty or condition of non-infringement, title, quality, merchantability or fitness for a particular purpose.6. Limitation of Liability In no event shall End User be entitled to recover from InSphero any special, indirect, incidental, consequential or punitive damages (including loss of profit, opportunity, business, savings, or reputation) in connection with this agreement or the license granted hereunder.7. Governing Law and Venue This Licence Agreement shall be governed by, and construed and enforced in accordance with the laws of Switzerland. Any action or proceeding arising from or relating to this License Agreement shall be brought in the Courts of Zurich, Switzerland and each party23InSphero AGWagistrasse 27CH-8952 SchlierenSwitzerlandPhone: +41 44 515 04 90e-mail:********************InSphero Inc.Brunswick, ME, USAPhone: +1 800-779-7558InSphero Europe GmbHWaldshut, GermanyPhone: +49 7751 3049665-0© 2015, InSphero AG, Switzerland.GravityTRAP™, GravityPLUS™, and SureXchange™ are trademarks ofInSphero AG. CellTiter-Glo® is a registered trademark of Promega Corp.All technical information subject to change.All products are for research use only. They are not intended for anyanimal or human therapeutic or diagnostic use unless otherwise stated.。