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InstantOne™ ELISA 384 Well Test ManualDoc. Part No. ELISA05677-1 Pub. No. MAN0017747 Rev.A.0WARNING! Read the Safety Data Sheets (SDSs) and follow thehandling instructions. Wear appropriate protective eyewear,clothing, and gloves. Safety Data Sheets (SDSs) are availablefrom /support.Assay principleInstantOne™ ELISA assays use the traditional sandwich ELISA format, but with a major difference. InstantOne™ ELISA allows for greater flexibility, ease of use, and reduced assay time by allowing the target analyte to bind to both of the two sandwich ELISA antibodies in solution as the capture antibody binds to the plate through a proprietary mechanism. This allows for both the sample and the assay reagents to be added to the InstantOne™ ELISA assay microplate at the same time. Unbound assay reagents and nonspecific sample components are washed away just as in a traditional sandwich ELISA, while the specific analyte is detected though a colorimetric detection reagent. The whole process can take just over 60 minutes to complete. In addition to the ease that the 1-hour/1-wash InstantOne™ ELISA provides, it also adds a layer of flexibility not readily accessible with traditional sandwich ELISAs. As the antibodies are not precoated in the wells, several different targets can be analyzed simultaneously in the same plate in different wells. Simply add the sample lysate to the plate wells and add different antibody cocktails to the different wells. It has never been easier to analyze both total and phosphorylated MAP Kinase family members or across pathways (e.g., ERK and AKT) in the same plate.InstantOne™ ELISA assay overview InstantOne™ ELISA assayworkflowInstantOne™ ELISA assay protocolworkflowTarget overview MAP kinase familyAKT pathwayAssay kit components and storageComponentsReagents and wells for 384 reactionsQuantity:Sufficient reagents and wells to perform 384 reactions per kitAlternatively: 96 well plate formats are available. Additional Wash Buffer (10X) and Cell Lysis Buffer (5X) are also available. Contact Technical Support for further information.•InstantOne ELISA Assay Plate: 10, 25, or 50 384-well platesspecifically designed and manufactured for this assay. Use onlyInstantOne™ ELISA Assay plates for InstantOne™ ELISA ELISAs.The plate is specifically designed to work with this assay andcannot be substituted with other 384-well microplates. Platesshould be stored at 2-8°C. Allow plate to equilibrate to roomtemperature prior to opening the pouch, to minimize condensation from forming in the wells. Unused wells should be stored dry at 2–8°C and used within 1 month of opening the microplate foil bag.Note: Nonstrip-well format and 384-well versions of the plate are available for special purchase. Contact Technical Support for further information.•Cell Lysis Buffer: The Cell Lysis Mix is a combination of the Cell Lysis Buffer and Enhancer Solution. The Cell Lysis Buffer (5X) contains a combination of detergents, phosphatase inhibitors, salts, and buffers. Cell Lysis Buffer (5X) is supplemented with Enhancer Solution to yield a versatile Cell Lysis Mix that can be applied to many cells and tissues. Note the difference in names. Cell Lysis Mix is referred to heavily in the assay protocol.–The Cell Lysis Mix (5X) is used to lyse cells in the presence ofculture medium and is typically used to lyse non-adherent cells.–The Cell Lysis Mix (1X) is prepared by simply diluting CellLysis Mix (5X) (a mixture of the Cell Lysis Buffer (5X) and theEnhancer Solution) 5-fold with water. This buffer is used to lyse cells after the removal of culture medium, and is typically used to lyse adherent cells or non-adherent cells that have beenharvested by centrifugation. Cell Lysis Mix (1X) should be used as the diluent for any dilution of cellular lysates that arerequired.Note: Supplementing Cell Lysis Mix with extra components (e.g., protease inhibitors, chelating agents, detergents) should be tested on a case-by-case basis for compatibility with InstantOne™ ELISA assays.•Wash Buffer (10X): The Wash Buffer, supplied as a 10X concentrate, is used for washing the InstantOne™ ELISA assay microplate. It is a simple mix of buffer, salts, and mild detergent. Alternatively, a PBS, 0.05% (v/v) Tween™ 20 solution may be substituted as a wash solution. If washing wells with a microplate washer, use 3X washes with a 10-second mixing cycle.•Detection Reagent: The emission filter should be in the range of 450 nm, with bandwidths ≤30 nm. The signal in the wells should be developed for around 15 minutes. Best results will be obtained if the microplates are developed in the dark (e.g., by covering the microplate with foil). It is recommended to protect the plate from light while undergoing development.•Stop Solution: The Stop Solution is used for stopping HRP-mediated colorimetric conversion. When added to the wells, the HRP enzyme activity stops and the detection reagent turns from blue to yellow with deeper yellow indicating a higherconcentration of target over a lighter development. The plateshould be read immediately after the addition of the stop solution.WARNING! Take caution because the Stop Solution is acid.Assay target specific reagents•Capture Antibody Reagent (Part No. Kit Specific): Contains the Capture Antibody Reagent that will be mixed in equal parts to the Detection Antibody Reagent to yield the Antibody Cocktail (ELISA antibody sandwich pair).•Detection Antibody Reagent (Part No. Kit Specific): Contains the Detection Antibody Reagent that will be mixed in equal parts to the Capture Antibody Reagent to yield the Antibody Cocktail (ELISA antibody sandwich pair). The Antibody Cocktail can be prepared by adding an equal volume of Capture Antibody Reagent and Detection Antibody Reagent, and mixing by inversion prior to each experiment.•Positive Control Cell Lysate (Part No. Kit Specific): Positive Control Cell Lysate is prepared from various cell types, which have been cultured and prepared to optimize the activation of the intracellular pathway of interest.–The Positive Control Cell Lysate is intended for use as an assay positive control only, and should not be used for the absolute quantification of a particular protein or phosphorylated target.In combination with negative control wells containing CellLysis Mix (1X) only, the Positive Control Cell Lysate can be used to give an indication of the expected signal range for a given assay.–The Positive Control Cell Lysate controls are suppliedlyophilized, and should be reconstituted with 250 µL of reagent grade dd H 2O. If required, Positive Control Cell Lysate can be further diluted with Cell Lysis Mix (1X), and frozen at less than –20°C in aliquots for subsequent use.Materials required but not supplied•Colorimetric plate reader capable of detecting 450 nm •Multichannel pipet (optional)•Reagent grade waterStorage conditionsStore kit components at the temperatures indicated on the labels.When handled as described below, the kit is stable for 6 months from date of receipt.Store all reagents at 2 – 8°C. Do NOT freeze the kits.Assay preparationBuffer preparationNote: Avoid vortexing the Capture Antibody Reagent or Detection Antibody Reagent, as vigorous mixing can damage some antibodies.[1]Bring all reagents to room temperature before use.Assay protocolsSample preparationProtocol for adherent cultured cellsRemove any media from the cells and gently wash cells with PBS.1.For cells cultured in 96-well microplates, lyse the cells with 100 µL of freshly prepared Cell Lysis Buffer Mix (1X).Note: Lysis volume should be adjusted depending on the desired lysate concentration. Lysates in the range of 0.1-0.5 mg/mL protein are usually sufficient. However, preparing more concentrated lysates can help with the detection of low abundance analytes.2.Shake cells (~300 rpm) at room temp for 10 minutes.Protocol for non-adherent cells1.Centrifuge the cells, gently remove the media while leaving thecells undisturbed. It is recommended, but not required, to wash the cells in PBS. Resuspend the cell pellet at an appropriate density in HBSS containing 5% FBS. A cell density that yields cellular lysate at a protein concentration of 0.1 - 0.5 mg/mL is suitable for many cell lines.Note: Alternatively re-suspend cells in cell culture medium if necessary for the cells.2.Return cells to a 37°C incubator for 1-2 hours.Note: For certain pathways, this can allow handling-mediatedpathway activation to subside. This step is optional, and depends on the activation status of your cells following re-suspension. 3.At the completion of the treatment, lyse cells with 20% finalvolume of Cell Lysis Mix (5X), with shaking (~300 rpm) at roomtemp for 10 min (e.g. for 40 µL of cells, use 10 µL of Cell Lysis Mix (5X).4.Alternatively cells can be harvested by centrifugation and lysedwith Cell Lysis Mix (1X).Assay protocol1.Determine and remove the desired number of InstantOne™ ELISA384-well plates needed for the experiment.2.Addition of negative control, positive Control, and sample lysateto assay wellsa.Add 10 µL/well of prepared sample lysate (as described above)to be tested to each of the InstantOne™ ELISA assay wells.b.Add 10 µL/well of Cell Lysis Mix (1X) (negative control) and10 µL/well of Positive Control Cell Lysate to separate wells forassay controls. The negative control can also act as the blankwhen the plate is read.3.Add 10 µL/well of prepared Antibody Cocktail to each of thetesting wells. Cover the microplate with adhesive seal andincubate for 1 hr at room temp on a microplate shaker (~300 rpm).Note: Remove Detection reagent from refrigerator and allow toequilibrate to room temperature.4.Wash wells with 40 µL/well of Wash Buffer (1X) (repeat 3 times).After final wash, completely remove any remaining wash solution from wells by inverting on a paper towel.5.Add 20 µL of the Detection Reagent to each of the wells. Incubatefor 10-30 minutes with shaking at 300 rpm. Watch colordevelopment as high abundance targets/samples will takesignificantly less time than lower abundant targets.6.Stop the reaction by adding 20 µL of Stop Solution to each well.7.Read the plate by measuring the absorbance of the samples usinga colorimetric (spectrophotometric) plate reader set at 450 nm.Plate should be read within 1 hour of adding the Stop Solution. Data analysis•To analyze the data, calculate the averaged counts for untreatedand treated cells. It is recommended to run the assay at least induplicate wells (n = 2) to calculate a response, but triplicate isstrongly advised.•Dose response and dose inhibition curves can be fitted to 4parameter nonlinear regression equations. These types ofregression analyses output key parameters such as EC50 (or IC50), Min and Max signals, and Hillslope factors.•Ensure that samples readings are within the linear range of theassay. This can vary based on reader performance, and analyteconcentration. If a lysate sample generates a signal outside thelinear range, the lysate samples should be diluted with Cell Lysis Mix (1X) and re-assayed.Procedure limitations•InstantOne™ ELISA kits are for Research Use Only. Not for use in diagnostic procedures.•Do not use the kit reagents beyond the expiry stated on the label.•Variations in general operator-related procedures, such aspipetting, washing, and incubation times, can cause variation inthe final signal.•The assay is designed to work for the detection of endogenouscellular proteins across a wide variety of cell lines. However, until each cell line in particular is tested, the possibility of the presence of interfering factors cannot be excluded.•Users should ensure that their cell line has measurable levels of the pathway of interest. Expression levels of signaling proteins indifferent cell types vary widely.Technical hints and troubleshootingVisit /support for the latest in services and support, including:•Worldwide contact telephone numbers•Product support, including:–Product FAQs–Software, patches, and updates–Training for many applications and instruments•Order and web support•Product documentation, including:–User guides, manuals, and protocols–Certificates of Analysis–Safety Data Sheets (SDSs; also known as MSDSs)Note: For SDSs for reagents and chemicals from othermanufacturers, contact the manufacturer.Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of Sale found on Life Technologies' website at /us/en/home/global/terms-and-conditions.html. If you have any questions, please contact Life Technologies at /support.Corporate entity: Life Technologies Corporation | Carlsbad, CA 92008 USA | Toll Free in USA 1 800 955 6288The information in this guide is subject to change without notice.DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses.©2018 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. All other trademarks are the property of their respective owners./support | /askaquestion。
TUNEL 细胞凋亡检测试剂盒(显色法)产品编号 产品名称包装 C1098TUNEL 细胞凋亡检测试剂盒(显色法)50次产品简介：碧云天生产的显色法TUNEL 细胞凋亡检测试剂盒(Colorimetric TUNEL Apoptosis Assay Kit)为您提供了一种高灵敏度又快速简便的细胞凋亡检测方法。
对于待检测的细胞或组织样品，经过生物素标记和后续的DAB 显色等步骤，即可在普通光学显微镜下观察到凋亡细胞。
细胞在发生凋亡时，会激活一些DNA 内切酶，这些内切酶会切断核小体间的基因组DNA 。
细胞凋亡时抽提DNA 进行电泳检测，可以发现180-200bp 的DNA ladder 。
基因组DNA 断裂时，暴露的3'-OH 可以在末端脱氧核苷酸转移酶(Terminal Deoxynucleotidyl Transferase, TdT)的催化下加上生物素(Biotin)标记的dUTP(Biotin-dUTP)，随后和辣根过氧化物酶(HRP)标记的Streptavidin (Streptavidin-HRP)结合，最后在HRP 的催化下通过DAB 显色来显示凋亡细胞，从而可以通过普通光学显微镜检测到凋亡的细胞，这就是TUNEL(TdT-mediated dUTP Nick-End Labeling)法检测细胞凋亡的原理。
本试剂盒有如下优点。
(1) 高灵敏度：背景染色极低，阳性染色强，可以在单细胞水平检测到细胞凋亡，同时由于凋亡早期就有DNA 断裂，可以检测到早期的细胞凋亡。
(2) 特异性好：TUNEL 检测时通常更容易标记凋亡细胞，而不容易标记坏死细胞。
(3) 快速：仅需约2-3个小时即可完成。
(4) 应用范围广：可以用于检测冷冻或石蜡切片中的细胞凋亡情况，也可以检测培养的贴壁细胞或悬浮细胞的凋亡情况。
(5) 实测效果好：参考图1。
图1. 本试剂盒的检测效果图。
A. HeLa 细胞未处理或用DNase I 室温孵育10分钟后的检测效果图。
补骨脂素对人牙周膜干细胞增殖、成骨分化的促进作用及其机制韩敏，张韶君，席迅山东第一医科大学第一附属医院（山东省千佛山医院）口腔科，济南 250014摘要：目的 观察补骨脂素对人牙周膜干细胞（hPDLSCs ）增殖、成骨分化的促进作用，并探讨其机制。
方法 将第三代hPDLSCs 分为6组，5、10、25、50、100 μmol /L 补骨脂素组加入5、10、25、50、100 μmol /L 补骨脂素，对照组正常培养，采用CCK -8法检测细胞增殖能力，比色法检测碱性磷酸酶（ALP ）活性，茜素红染色检测成骨诱导矿化结节。
另取第三代hPDLSCs 并分为两组，25 μmol /L 补骨脂素组加入25 μmol /L 补骨脂素，对照组正常培养，采用RT -PCR 法检测转录因子2（Runx2）、骨桥蛋白（OPN ）mRNA 。
结果 与对照组比较，5、10、25、50、100 μmol /L 补骨脂素组细胞增殖能力和ALP 活性增加，25 μmol /L 补骨脂素组细胞成骨矿化结节增加（P 均<0.05）；与5 μmol /L 补骨脂素组比较，25、50、100 μmol /L 补骨脂素组细胞增殖能力及10、25 μmol /L 补骨脂素组细胞ALP 活性和25 μmol /L 补骨脂素组细胞成骨矿化结节增加（P 均<0.05）；与10 μmol /L 补骨脂素组比较，25、50、100 μmol /L 补骨脂素组细胞增殖能力增加（P 均<0.05）；与25 μmol /L 补骨脂素组比较，50、100 μmol /L 补骨脂素组细胞ALP 活性降低（P 均<0.05）。
与对照组比较，25 μmol /L 补骨脂素组Runx2、OPN mRNA 相对表达量增加（P 均<0.05）。
结论 5～100 μmol /L 补骨脂素均能促进hPDLSCs 的增殖和成骨分化，在5～25 μmol /L 呈剂量依赖性增强，在50～100 μmol /L 变化不明显；补骨脂素促进hPDLSCs 的增殖和成骨分化的机制可能与调节Runx2信号通路有关。
黑色素细胞抗体ELISA试剂盒说明书黑色素细胞抗体ELISA试剂盒说明书供应商：上海乔羽生物有限公司ELISA kit specification:(1) specifications: 96T can be measured 90 samples, 5 standard holes, 1 blank holes(2) specifications: 48T can be measured 42 samples, 5 standard holes, 1 blank holes黑色素细胞抗体ELISA试剂盒说明书预期应用：ELISA法定量测定人血清、血浆、细胞培养上清或其它相关生物液体中相关物质含量。
1．试剂盒保存：-20℃(较长时间不用时)；2-8℃(频繁使用时)。
2．浓洗涤液低温保存会有盐析出，稀释时可在水浴中加温助溶。
3．中、英文说明书可能会有不一致之处，请以英文说明书为准。
4．刚开启的酶联板孔中可能会含有少许水样物质，此为正常现象，不会对实验结果造成任何影响。
7. 不能检测含NaN3的样品，因NaN3抑制辣根过氧化物酶的（HRP）活性。
黑色素细胞抗体ELISA试剂盒说明书ELIAS试剂盒的实验条件:1.固相载体的选择：许多物质可作为固相载体，如聚氯乙烯、聚苯乙烯、聚丙酰胺和纤维素等。
其形式可以是凹孔平板、试管、珠粒等。
2. 包被抗体或抗原的选择：将抗体或抗原吸附在固相载体表面时，要求纯度要好,吸附时一般要求PH在9.0～9.6之间。
3.酶标记抗体工作浓度的选择：首先用直接ELISA法进行初步效价的滴定。
然后再固定其它条件或采取“方阵法"，在正式实验系统里准确地滴定其工作浓度。
4.酶的底物及供氢体的选择：对供氢体的选择要求是价廉、安全、有明显地显色反应，而本身无色。
有些供氢体有潜在的致癌作用，ELIAS试剂盒应注意防护。
黑色素细胞抗体ELISA试剂盒说明书Kit composition:Sealing film: 2 (48) / 2 (96)Specifications: 1 copiesSealing bag: 1Standard: 2700ng / L 0.5 * 0.5ml * 1 bottles, 1 bottles are stored in 2-8ELISA package is board: 1 X 48 x 961 2-8 stored inSample dilution: 3ml * 1 ml * 1 bottles of 6 bottles are stored in 2-8Color agent: Liquid 3ml * 1 ml * 6 bottles of 1 bottles in 2-8Color reagent B Liquid 3ml * 1 ml * 1 bottle 6 bottles stored in 2-8 Termination solution: 3ml * 6ml * 1 bottles and 1 bottles are stored in 2-8Concentrated laundry liquid: (2 * 20) * 1 bottles (20ml * 30) * 1 bottles stored in 2-8elisa试剂盒价格，elisa试剂盒说明书，elisa检测试剂盒黑色素细胞抗体ELISA试剂盒说明书试剂的准备：按试剂盒说明书的要求准备实验中需用的试剂。
人AKT蛋白（AKT）酶联免疫分析(ELISA)试剂盒使用说明书本试剂仅供研究使用目的：本试剂盒用于测定人血清、血浆、组织等样本中AKT蛋白（AKT）的含量。
实验原理：本试剂盒应用双抗体夹心法测定标本中人AKT蛋白（AKT）水平。
用纯化的人AKT蛋白（AKT）捕获抗体包被微孔板，制成固相抗体，往包被的微孔中依次加入人AKT蛋白（AKT），再与HRP标记的检测抗体结合，形成抗体-抗原-酶标抗体复合物，经过彻底洗涤后加底物TMB显色。
TMB在HRP酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。
颜色的深浅和样品中的人AKT蛋白（AKT）呈正相关。
用酶标仪在450nm波长下测定吸光度（OD值），通过标准曲线计算样品中人AKT蛋白（AKT）含量。
试剂盒组成：样本处理及要求：1. 血清：室温血液自然凝固10-20分钟，离心20分钟左右（2000-3000转/分）。
仔细收集上清，保存过程中如出现沉淀，应再次离心。
2. 血浆：应根据标本的要求选择EDTA或柠檬酸钠作为抗凝剂，混合10-20分钟后，离心20分钟左右（2000-3000转/分）。
仔细收集上清，保存过程中如有沉淀形成，应该再次离心。
3. 尿液：用无菌管收集，离心20分钟左右（2000-3000转/分）。
仔细收集上清，保存过程中如有沉淀形成，应再次离心。
胸腹水、脑脊液参照实行。
4. 细胞培养上清：检测分泌性的成份时，用无菌管收集。
离心20分钟左右（2000-3000转/分）。
仔细收集上清。
检测细胞内的成份时，用PBS（PH7.2-7.4）稀释细胞悬液，细胞浓度达到100万/ml左右。
通过反复冻融，以使细胞破坏并放出细胞内成份。
离心20分钟左右（2000-3000转/分）。
仔细收集上清。
保存过程中如有沉淀形成，应再次离心。
5. 组织标本：切割标本后，称取重量。
加入一定量的PBS，PH7.4。
用液氮迅速冷冻保存备用。
标本融化后仍然保持2-8℃的温度。
腺苷酸激酶5(AK5)ELISA试剂盒说明书本试剂仅供研究使用标本：体液腺苷酸激酶5(AK5)ELISA试剂盒说明书试验原理：BFGF试剂盒是固相夹心法酶联免疫吸附实验（ELISA）.已知BFGF浓度的标准品、未知浓度的样品加入微孔酶标板内进行检测。
先将BFGF和生物素标记的抗体同时温育。
人碱性成纤维细胞生长因子ELISA 检测试剂盒洗涤后，加入亲和素标记过的HRP。
再经过温育和洗涤，去除未结合的酶结合物，然后加入底物A、B，和酶结合物同时作用。
产生颜色。
颜色的深浅和样品中BFGF的浓度呈比例关系。
腺苷酸激酶5(AK5)ELISA试剂盒说明书自备材料1．蒸馏水。
2．加样器：5ul、10ul、50ul、100ul、200、500ul、1000ul。
3．振荡器及磁力搅拌器等。
安全性1．避免直接接触终止液和底物A、B。
一旦接触到这些液体，请尽快用水冲洗。
2．实验中不要吃喝、抽烟或使用化妆品。
3．不要用嘴吸取试剂盒里的任何成份。
腺苷酸激酶5(AK5)ELISA试剂盒说明书操作注意事项1．试剂应按标签储存，使用前恢复到室温。
稀稀过后的标准品应丢弃，不可保存。
2．实验中不用的板条应立即放回包装袋中，密封保存，以免变质。
3．不用的其它试剂应包装好或盖好。
不同批号的试剂不要混用。
保质前使用。
4．使用一次性的吸头以免交叉污染，吸取终止液和底物A、B液时，避免使用带金属部分的加样器。
5．使用干净的塑料容器配置洗涤液。
使用前充分混匀试剂盒里的各种成份及样品。
6．洗涤酶标板时应充分拍干，不要将吸水纸直接放入酶标反应孔中吸水。
7．底物A应挥发，避免长时间打开盖子。
底物B对光敏感，避免长时间暴露于光下。
避免用手接触，有毒。
实验完成后应立即读取OD值。
8．加入试剂的顺序应一致，以保证所有反应板孔温育的时间一样。
9．按照中标明的时间、加液的量及顺序进行温育操作。
腺苷酸激酶5(AK5)ELISA试剂盒说明书样品收集、处理及保存方法1、血清-----操作过程中避免任何细胞刺激。
美国贝克曼库尔特流式细胞分析仪（Beckman coulter cell）产品型号：Cell Lab Quanta SC当前价格：0.00元产品数量：0新旧程度：全新有效期至：0000-00-00所在地：产品简介：仪器简介：T细胞亚群检测的CD45/CD4/CD8/CD3、CD45/CD56/CD19/CD3；阵发性血红蛋白尿（PNH）检测的CD55、CD59；血小板无力症（GT）检测的CD41、CD61等等详细信息仪器简介：T细胞亚群检测的CD45/CD4/CD8/CD3、CD45/CD56/CD19/CD3；阵发性血红蛋白尿（PNH）检测的CD55、CD59；血小板无力症（GT）检测的CD41、CD61等等。
但对于白血病/淋巴瘤免疫分型，国际上迄今为止也没有统一的抗体组合。
在2000年国际细胞分析学会（ISAC）大会上，临床血细胞计数协会组织了一次国际专家会议，以期对检测血液淋巴系统肿瘤所需最少、最有效的单抗数达成共识。
75%与会者一致认为，对于慢性淋巴系统增殖性疾病（CLD）有9种单抗：CD5,CD19,κ,λ,CD3,CD20,CD23,CD10,CD45对初诊来说是最基本的。
淋巴瘤和CLD相似，需要至少12-16种单抗。
对于急性白血病（AL），75%的与会者认为大约13-15种单抗是最基本的：CD10,CD19,CD79a,CD13,CD33,CD34,CD45,CD2,MPO，CD7,CD14,CD3,HLA-DR等，对初步鉴别白血病系列是必需的。
其他一些（CD16,CD56,CDw65,TdT,cyCD3）可能对某些病例有用。
几乎所有的投票者都认为，要对急性白血病完善分类所需单抗的恰当数量平均为20-24种。
但这些抗体之间组合也是一大难题，目前也无统一规定（如表二）。
大会多数发言者(11/13)指出，对已确诊病人的监护和分期来说，仅需较少单抗。
抗体的质量控制是实验的关键环节。
抗体的质量包括其特异性、灵敏度、精密度。
（本试剂盒仅供体外研究使用，不用于临床诊断！）产品货号：E-EL-M3062产品规格：96T/48T/24T/96T*5Elabscience®小鼠白介素12(IL-12)酶联免疫吸附测定试剂盒使用说明书Mouse IL-12(Interleukin 12) ELISA Kit使用前请仔细阅读说明书。
如果有任何问题，请通过以下方式联系我们：销售部电话************，************技术部电话************具体保质期请见试剂盒外包装标签。
请在保质期内使用试剂盒。
联系时请提供产品批号(见试剂盒标签)，以便我们更高效地为您服务。
用途该试剂盒用于体外定量检测小鼠血清、血浆或其它相关生物液体中IL-12浓度。
检测原理本试剂盒采用双抗体夹心ELISA法。
用抗小鼠IL-12抗体包被于酶标板上，实验时样品（或标准品）中的小鼠IL-12会与包被抗体结合。
后依次加入生物素化的抗小鼠IL-12抗体和辣根过氧化物酶标记的亲和素，抗小鼠IL-12抗体与结合在包被抗体上的小鼠IL-12结合，生物素与亲和素特异性结合而形成免疫复合物，游离的成分被洗去。
加入显色底物(TMB)，TMB在辣根过氧化物酶的催化下呈现蓝色，加终止液后变成黄色。
用酶标仪在450 nm波长处测OD值，IL-12浓度与OD450值之间呈正比，通过绘制标准曲线计算出样品中IL-12的浓度。
试剂盒组成及保存未拆封的试剂盒可在2-8℃保存一周；如果一周以后才使用试剂盒，请拆开试剂盒并按照下表中的条件分别保存各组分。
试剂体积以实际发货版说明书为准。
相关试剂在分装时会比标签上试验所需自备物品1.酶标仪(450 nm波长滤光片)2.高精度移液器，EP管及一次性吸头：0.5-10μL, 2-20μL, 20-200μL, 200-1000μL3.37℃恒温箱，4.双蒸水或去离子水5.吸水纸6.加样槽样品收集方法(具体处理方法可参考官网：/List-detail-241.html) 1.血清：全血样品于室温放置1小时或2-8℃过夜后于2-8℃，1000×g离心20分钟，取上清即可检测。
（本试剂盒仅供体外研究使用，不用于临床诊断！）Elabscience®总胆固醇(TC)比色法测试盒(单试剂COD-PAP法)Total Cholesterol (TC) Colorimetric Assay Kit (Single Reagent, COD-PAP Method)产品货号：E-BC-K109-M产品规格：48T(44 samples)/96T(92 samples)检测仪器：酶标仪(495-525 nm)使用前请仔细阅读说明书。
如果有任何问题，请通过以下方式联系我们：销售部电话************，************技术部电话131****6790具体保质期请见试剂盒外包装标签。
请在保质期内使用试剂盒。
联系时请提供产品批号(见试剂盒标签)，以便我们更高效地为您服务。
用途本试剂盒适用于检测血清（浆）、组织的总胆固醇含量。
检测原理总胆固醇(Total Cholesterol，TC)包括游离胆固醇和胆固醇酯。
胆固醇酯可被胆固醇酯酶（cholesterol esterase，CE）水解成胆固醇和游离脂肪酸，胆固醇在胆固醇氧化酶（cholesterol oxidase，CO）的氧化作用下生成△4-胆甾烯酮和过氧化氢。
过氧化氢在4-氨基安替吡啉和酚存在时，经过氧化物酶（peroxidase，POD）催化，反应生成苯醌亚胺非那腙的红色醌类化合物，其颜色深浅与TC含量成正比。
其检测原理如下图：提供试剂和物品说明：试剂严格按上表中的保存条件保存，不同测试盒中的试剂不能混用。
对于体积较少的试剂，使用前请先离心，以免量取不到足够量的试剂。
所需自备物品仪器：酶标仪（495-525 nm）、微量移液器（1000 μL，200 μL，100 μL，10 μL）、恒温箱、离心机。
耗材：枪头（1000 μL、200 μL、2.5 μL）、EP管（2 mL）。
试剂：双蒸水、生理盐水（0.9% NaCl）或PBS（0.01 M，pH 7.4）、无水乙醇。
罗氏公司TUNEL细胞凋亡检测程序（In situ cell death detection kit-POD法）一、原理：TUNEL（TdT-mediated dUTP nick end labeling）细胞凋亡检测试剂盒是用来检测组织细胞在凋亡早期过程中细胞核DNA的断裂情况。
其原理是荧光素（fluorescein）标记的dUTP在脱氧核糖核苷酸末端转移酶（TdT Enzyme）的作用下，可以连接到凋亡细胞中断裂DNA的3’-OH末端，并与连接辣根过氧化酶（HRP，horse-radish peroxidase）的荧光素抗体特异性结合，后者又与HRP底物二氨基联苯胺（DAB）反应产生很强的颜色反应（呈深棕色），特异准确地定位正在凋亡的细胞，因而在光学显微镜下即可观察凋亡细胞；由于正常的或正在增殖的细胞几乎没有DNA断裂，因而没有3'-OH形成，很少能够被染色。
本试剂盒适用于组织样本（石蜡包埋、冰冻和超薄切片）和细胞样本（细胞涂片）在单细胞水平上的凋亡原位检测。
还可应用于抗肿瘤药的药效评价，以及通过双色法确定细胞死亡类型和分化阶段。
二、器材与试剂器材：光学显微镜及其成像系统、小型染色缸、湿盒（塑料饭盒与纱布）、塑料盖玻片或封口膜、吸管、各种规格的加样器及枪头等；试剂：试剂盒含TdT 10×、荧光素标记的dUTP 1×、标记荧光素抗体的HRP；自备试剂：PBS、双蒸水、二甲苯、梯度乙醇（100、95、90、80、70％）、DAB工作液（临用前配制，5 µl 20×DAB＋1μL30％H2O2＋94 µl PBS）、Proteinase K工作液（10-20 μg/ml in 10 mM Tris/HCl, pH 7.4-8）或细胞通透液（0.1% Triton X-100 in 0.1% sodium citrate，临用前配制）、苏木素或甲基绿、DNase 1（3000 U/ml– 3 U/ml in 50 mM Tris-HCl，pH 7.5, 10 mM MgCl2，1 mg/ml BSA）等。
INSTRUCTIONSPierce Colorimetric In-Cell ELISA Kits62200 In-Cell ELISA Colorimetric Detection Kit, sufficient materials for 4 × 96 wells62205 Pierce EGFR Colorimetric In-Cell ELISA Kit, sufficient materials for 1 × 96 wells62206 Pierce ERK1/2 Colorimetric In-Cell ELISA Kit, sufficient materials for 1 × 96 wells62207 Pierce S6 Colorimetric In-Cell ELISA Kit, sufficient materials for 1 × 96 wells62208 Pierce STAT6 Colorimetric In-Cell ELISA Kit, sufficient materials for 1 × 96 wells62209 Pierce STAT3 Colorimetric In-Cell ELISA Kit, sufficient materials for 1 × 96 wells62215 Pierce AKT Colorimetric In-Cell ELISA Kit, sufficient materials for 1 × 96 wells62216 Pierce p53 Colorimetric In-Cell ELISA Kit, sufficient materials for 1 × 96 wells62217 Pierce GSK3 α/β Colorimetric In-Cell ELISA Kit, sufficient materials for 1 × 96 wells62218 Pierce Cleaved Caspase 3 Colorimetric In-Cell ELISA Kit, sufficient materials for 1 × 96 wells 62219 Pierce Cleaved PARP Colorimetric In-Cell ELISA Kit, sufficient materials for 1 × 96 wellsKit ContentsBlocking Buffer, 50mL20X Tris Buffered Saline, 50mLSurfact-Amps 20 (10% Tween™ 20 Detergent), 10mLSurfact-Amps X-100 (10% Triton™ X-100 Detergent), 10mLHRP Conjugate, 0.14mLTMB Substrate, 58mLTMB Stop Solution, 55mLJanus Green Whole-Cell Stain, 50mLElution Buffer, 2 × 30mLThin Plate Seal Assembly, 8 eachComponents included only in the target-specific kits:Antibody #1, see vial labelAntibody #2, see vial labelStorage: Upon receipt, store all components except antibodies at 4°C. Store the antibodies at temperatures indicated on the antibody vial. Allow buffers to warm to room temperature before use. See the Solution Preparation Section for storage and stability of prepared solutions. Kit is shipped with an ice pack.Warning: Completely read these instructions and the accompanying material safety data sheets before using this product. Reagents provided are not for diagnostic use in humans or animals.IntroductionThe Thermo Scientific™ Pierce™ In-Cell ELISA Colorimetric Detection Kit is a simple and convenient method for quantifying intracellular proteins in whole cells. To perform the assay, cells are first plated, treated and fixed. Expression of the protein(s) of interest is monitored in wells of a microplate using target-specific primary antibodies (see the Important Product Information section for antibodies included in each kit) and a horseradish peroxidase (HRP)-conjugated detection reagent. The kit is supplied with a whole-cell stain to control for differences in cell plating, which is important when measuring relative levels of a protein with different treatments or assessing its post-translational modification (PTM) form. After staining, the results are analyzed by normalizing the absorbance (HRP activity) values to cell number, which adjusts for the cell plating differences among the wells.Traditionally, relative protein levels in various samples or PTMs were assessed by performing time-consuming Western blots, which are semi-quantitative and have low throughput. In contrast, the in-cell ELISA method enables accurate quantitation using a standard ELISA plate reader. The assay is performed in a 96- or 384-well microplate, is scalable, and conserves cell culture and treatment reagents. Furthermore, the assay is amenable to automation, which is ideal for siRNA studies and drug screens.Important Product Information•The Pierce In-Cell ELISA Kits are offered in two formats: without primary antibodies (Product No. 62200) or with target-specific antibodies (Product No. 62205-62209; 62215-62219).•The HRP Conjugate that is included in each kit will recognize and bind primary antibodies (IgG) from several species, including mouse and rabbit IgG.•Each target-specific kit contains two antibodies (Table 1). Please see our web site for detailed background information for each target and kit-specific data.Table 1. Target-specific antibodies included in each kit.Product # Target Antibodies Included62205 EGFR #1: Anti-Phospho EGFR (Y1173) Antibody #2: Anti-EGFR Antibody62206 ERK1/2 #1: Anti-ERK 1 & 2 (Thr 202/Tyr 204) Antibody #2: Anti-ERK1/2 Antibody62207 S6 #1: Anti-Phospho S6 (Ser 235/236) Antibody #2: Anti-S6 Antibody62208 STAT6 #1: Anti-Phospho STAT6 (Y641) Antibody #2: Anti-STAT6 Antibody62209 STAT3 #1: Anti-Phospho STAT3 (Y705) Antibody #2: Anti-STAT3 Antibody62215 AKT #1: Anti-Phospho AKT (S473) Antibody #2: Anti-AKT Antibody62216 p53 #1: Anti-p53 Antibody#2: Anti-Alpha Tubulin Antibody62217 GSK3 α/β#1: Anti-Phospho GSK3 a/b (S21/9) Antibody #2: Anti-GSK3 Antibody62218 Cleaved Caspase 3 #1: Anti-Cleaved Caspase 3 Antibody #2: Anti-Alpha Tubulin Antibody62219 Cleaved PARP #1: Anti-Cleaved PARP Antibody #2: Anti-Alpha Tubulin AntibodyProcedure Summary1.Prepare plates (i.e., plate and treat cells as desired, and then fix them using 4% PFA).2.Permeabilize, quench endogenous peroxidase and block nonspecific sites with blocking buffer.3.Detect targets with primary antibodies and the HRP-conjugate.4.Measure absorbance at 450nm.5.Stain cells with whole-cell stain (steps 5-7 are optional).6.Elute whole-cell stain.7.Measure absorbance at 615nm.8.Analyze results.Additional Materials Required•Disposable reagent reservoirs (Thermo Scientific ImmunoWare Reagent Reservoirs, Product No. 15075)•Standard ELISA reader for measuring absorbance at 450nm and 615nm•Methanol-free formaldehyde (Thermo Scientific 16% Formaldehyde, Product No. 28906), diluted to 4%•30% hydrogen peroxide (H2O2,Sigma Product No. H1009), diluted to 1%•96-well cell culture clear-bottom microplates, such as black collagen-coated plates (Nunc, Product No. 152036), clear collagen-coated plates, (BD, Product No. 354407), black clear-bottom plates, (Perkin-Elmer, Product No. 6005182) or clear plates (Corning, Product No. 3596)Precautions•All samples and reagents must be at room temperature (20-25°C) before use in the assay.•To avoid cross-contamination use new disposable pipette tips for each transfer and a new adhesive plate cover for each antibody incubation step. If using a multichannel pipette, always use a new disposable reagent reservoir.•Take care not to let plate dry at any time during the assay.•Avoid exposing reagents to excessive heat or light during storage and incubation.•Do not mix reagents from different kit lots, and discard unused ELISA components after assay completion. Do not combine leftover reagents with those reserved for additional plates.•Do not use glass pipettes to measure the Substrate Solution. Take care not to contaminate the solution. If solution is blue before use, do not use it.•Individual components may contain antibiotics and preservatives. Wear gloves while performing the assay to avoid contact with samples and reagents. Please follow proper disposal procedures.•Dispense and equilibrate to room temperature only the reagent volumes required for the number of plates being used. •Briefly centrifuge the tubes of primary antibody before use.In-Cell ELISA Protocol•Perform all incubations with gentle shaking on a plate shaker.•To remove the plate contents, rapidly invert the plate over a waste receptacle. Tap the inverted plate gently three times on a paper towel or other absorbent material to remove any remaining solution.•Perform each wash step for 5 minutes with gentle shaking on a plate shaker.A.Solution Preparation (per 96-well plate)1X Tris Buffered Saline (TBS) Add 2.5mL of 20X TBS to 47.5mL of ultrapure water. Store buffer at 4°C for up to7 days.4% Formaldehyde Add 2.75mL of 16% methanol-free formaldehyde to 8.25mL of 1X TBS. Preparesolution just before each assay.1X Permeabilization Buffer Add 0.11mL of Thermo Scientific™ Surfact-Amps™ X-100 Detergent to 11mL of the1X TBS. Store this buffer at 4°C for up to 7 days.Quenching Solution Add 0.38mL of 30% H2O2 to 11mL of 1X TBS to make 1% H2O2. Prepare solutionjust before each assay.1X Wash Buffer Add 7.5mL of 20X TBS to 141mL of ultrapure water. Add 1.5mL of Surfact-Amps 20Detergent. Store buffer at 4°C for up to 7 days.Diluted Primary Antibody Add 3 ml of Blocking Buffer to 3mL of 1X Wash Buffer. Dilute the primary antibodywith this solution to the dilution stated on the antibody vial.* This volume is sufficientfor one target antibody added to 96 wells. Adjust the volume of the antibody solutionbased on the number of targets and wells being tested.Diluted HRP Conjugate Add 30µL of HRP Conjugate to 12mL of 1X Wash Buffer. Prepare solution just beforeeach assay.*If using Product No. 62200, dilute the antibody as indicated by the supplier.B.Assay Procedure1.Plate 10,000 cells/well in a 96-well plate. Incubate plates overnight at 37°C in 5% CO2. Use only cells growing in logphase at a passage number ≤ 15.Note: Plate enough wells to perform the experiment in triplicate. Include appropriate controls such as nonspecific signal(i.e., wells treated with all reagents except the primary antibody).2.Apply cell treatment as necessary.3.Remove the media and add 100µL of 4% formaldehyde to each well. Incubate the plate in a fume hood at roomtemperature for 15 minutes.Note: Formaldehyde and its vapors are highly toxic. Perform steps involving formaldehyde in a fume hood. Discard the formaldehyde waste according to your local regulations.4.Remove formaldehyde and wash plate twice with 100µL/well of 1X TBS.5.Remove 1X TBS, add 100µL/well of 1X Permeabilization Buffer and incubate for 15 minutes at room temperature.6.Remove Permeabilization Buffer and wash plate once with 100µL/well of 1X TBS.7.Remove 1X TBS, add 100µL/well of Quenching Solution and incubate at room temperature for 20 minutes.8.Remove Quenching Solution and wash plate once with 100µL/well of 1X TBS.9.Remove 1X TBS, add 100µL/well of Blocking Buffer and incubate at room temperature for 30 minutes.10.Remove Blocking Buffer and add 50µL/well of primary antibody. Apply a plate sealer and incubate overnight at 4°C.11.Remove the primary antibody solution and wash plate three times with 100µL/well of 1X Wash Buffer.12.Remove Wash Buffer and add 100µL/well of Diluted HRP Conjugate. Incubate for 30 minutes at room temperature.13.Remove the Diluted HRP Conjugate and wash plate three times with 200µL/well of 1X Wash Buffer.14.Remove Wash Buffer and add 100µL/well of TMB Substrate. Incubate at room temperature protected from light. Stopthe reaction within 15 minutes or when the desired blue color has been achieved.Note: Dispense from bottle only the amount of reagent required for the number of wells being used. Do not use a glass pipette to measure the TMB Substrate. Take care not to contaminate remaining substrate solution.15.Add 100µL/well of TMB Stop Solution. Measure the absorbance at 450nm (A450) within 30 minutes of stopping the reaction.C.Whole Cell Staining (optional)1.Empty the plate contents and wash plate twice with 200µL/well of ultrapure water.2.Remove water and add 100µL/well of Janus Green Whole-Cell Stain. Incubate plate for 5 minutes at room temperature.3.Remove stain and wash with 3-5 times with 200µL/well of ultrapure water until all excess stain is removed.4.Remove water and add 100µL/well of the Elution Buffer. Incubate for 10 minutes at room temperature. Measure theabsorbance at 615nm (A615).D.Results Calculation1.Calculate the average of all replicate background measurements (nonspecific signal control) from each experimentalcondition.2.Subtract the background values from all values of the same experimental type.3.If Janus green staining was performed, normalize the A450 values to A615 values from corresponding wells to account fordifferences in cell numbers in various wells by dividing the net A450 values by the A615 values.4.Calculate the average A450 value for each experimental condition (e.g., with and without treatment) for each target. Forassessing target protein modification with treatment, calculate the fold change as a ratio of the treated and nontreated modified protein A450 values.Note: To compensate for changes in total target protein levels with treatment (irrespective of PTM) measure the levels of non-modified target protein using the same experimental conditions. Normalize the average A450 value obtained for the modified protein to that of the average A450 value for the total target protein. Use the normalized A450 values to calculate the fold change.Data TemplateDate: Cell Type:TroubleshootingAdditional InformationVisit our web site for additional information relating to this product including the following: •Target-specific data•Application notes and referencesRelated Thermo Scientific Products62200 In-Cell ELISA Colorimetric Detection Kit62201 In-Cell ELISA Near-Infrared Fluorescence Detection Kit62203 Janus Green Whole Cell Stain, 50mL62204 Anti-α-Tubulin Antibody, 100µL (200µg/mL)34028 1-Step Ultra TMB-ELISA, 250mL28358 20X TBS Buffer, 500mL28320 Surfact-Amps 20 (10% Tween-20 Detergent), 6 × 10mL28314 Surfact-Amps X-100 (10% Triton X-100 Detergent), 6 × 10mLProducts are warranted to operate or perform substantially in conformance with published Product specifications in effect at the time of sale, as set forth in the Product documentation, specifications and/or accompanying package inserts (“Documentation”). No claim of suitability for use in applications regulated by FDA is made. The warranty provided herein is valid only when used by properly trained individuals. Unless otherwise stated in the Documentation, this warranty is limited to one year from date of shipment when the Product is subjected to normal, proper and intended usage. This warranty does not extend to anyone other than Buyer. Any model or sample furnished to Buyer is merely illustrative of the general type and quality of goods and does not represent that any Product will conform to such model or sample.NO OTHER WARRANTIES, EXPRESS OR IMPLIED, ARE GRANTED, INCLUDING WITHOUT LIMITATION, IMPLIED WARRANTIES OF MERCHANTABILITY, FITNESS FOR ANY PARTICULAR PURPOSE, OR NON INFRINGEMENT. BUYER’S EXCLUSIVE REMEDY FOR NON-CONFORMING PRODUCTS DURING THE WARRANTY PERIOD IS LIMITED TO REPAIR, REPLACEMENT OF OR REFUND FOR THE NON-CONFORMING PRODUCT(S) AT SELLER’S SOLE OPTION. THERE IS NO OBLIGATION TO REPAIR, REPLACE OR REFUND FOR PRODUCTS AS THE RESULT OF (I) ACCIDENT, DISASTER OR EVENT OF FORCE MAJEURE, (II) MISUSE, FAULT OR NEGLIGENCE OF OR BY BUYER, (III) USE OF THE PRODUCTS IN A MANNER FOR WHICH THEY WERE NOT DESIGNED, OR (IV) IMPROPER STORAGE AND HANDLING OF THE PRODUCTS.Unless otherwise expressly stated on the Product or in the documentation accompanying the Product, the Product is intended for research only and is not to be used for any other purpose, including without limitation, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses, or any type of consumption by or application to humans or animals.Current product instructions are available at /pierce. For a faxed copy, call 800-874-3723 or contact your local distributor.© 2014 Thermo Fisher Scientific Inc. All rights reserved. Tween is a trademark of Croda International Plc. Triton is a trademark of Union Carbide Corp. Unless otherwise indicated, all other trademarks are property of Thermo Fisher Scientific Inc. and its subsidiaries. Printed in the USA.。
