磁珠M-280使用说明

AMMS TM CD3/CD28抗体偶联磁珠说明书产品名称通用名称：CD3/CD28抗体偶联磁珠英文名称：Anti-human CD3/CD28 monoclonal antibody beads适用范围AMMS TM CD3/CD28磁珠适用于人T细胞的分离，激活和扩增。

适用于直肠癌、乳腺癌、肺癌、肾癌、淋巴瘤、白血病、多发性骨髓瘤、恶性黑色素瘤、卵巢癌等多种肿瘤的细胞免疫治疗临床研究。

AMMS CD3/CD28磁珠提供了一种不需要抗原呈递细胞和抗原就能激活和扩增调节T细胞的简单的方法。

通过在磁珠上组合抗CD3和抗CD28抗体，就能提供调节T细胞激活和扩增需要的初级和协同刺激信号。

使用说明一、AMMS TM CD3/CD28磁珠的清洗：1.1 在小管里重悬磁珠（也就是说，涡旋超过30s，或者颠倒混匀5min）1.2 将确定体积的磁珠转移到管子中。

1.3 加入等体积的PBS缓冲液含1%的HAS，或者至少1ml的体积，进行重悬。

1.4把管子放在一个磁铁上1min，随后弃去上清。

1.5将管子从磁铁上转移下来，用相同体积的PBS缓冲液含1%的HAS重悬磁珠（第二步最初体积的磁珠）。

二、磁珠分离和扩增CD3+的T细胞注意：由于单核细胞在37℃能够快速的吞噬磁珠，因此就降低了所能够接触到T细胞磁珠的绝对数量，进而降低了T细胞活化和扩增的能力。

2.1 通过流式或者是其他的方法确定样本中CD3+T细胞的比例。

2.2 对于Ficoll分离的PBMC，将细胞轻柔的重悬于含1%HAS的PBS缓冲液中，并调节细胞密度为2-5×107个/mL，此处注意，总的细胞数量不要超过2×108个。

2.3 将磁珠和细胞比例为3:1，加入已经洗过的AMMS TM CD3/CD28磁珠。

2.4 将样品置于1转的摇床上，在4℃-25℃条件下孵育30min，在每次的试验中需要在4℃-25℃范围内优化最适的孵育温度。

2.5用无血清培养基或者含1%HAS的PBS缓冲液稀释磁珠和细胞的混合物，来保证磁铁的分选体积。

磁珠法核酸提取试剂盒说明书磁珠法核酸提取试剂盒说明书在科学研究和临床诊断中，核酸提取是至关重要的一步。

随着技术的不断发展，磁珠法核酸提取试剂盒逐渐成为了研究人员和临床医生们的首选。

那么，什么是磁珠法核酸提取试剂盒？它的工作原理是什么？在使用过程中需要注意哪些事项？本文将从广度和深度两方面进行全面评估，带你深入了解磁珠法核酸提取试剂盒。

一、磁珠法核酸提取试剂盒的工作原理磁珠法核酸提取试剂盒是利用磁珠的磁性特性，结合核酸和其他杂质的不同特性，通过磁场的作用将目标核酸分离提取出来的一种试剂盒。

具体步骤包括细胞裂解、磁珠与核酸结合、洗涤和最终洗脱等过程。

通过这一系列步骤，能够快速、高效地从样本中提取出纯度较高的核酸，为后续的分子生物学实验和临床检测打下基础。

磁珠法核酸提取试剂盒本质上是一种高度精密的技术产品，涉及到多方面的知识和技术。

在使用过程中需要严格按照说明书上的操作步骤进行，以确保提取的核酸具有良好的质量。

二、磁珠法核酸提取试剂盒的使用注意事项在使用磁珠法核酸提取试剂盒时，有一些注意事项是需要特别关注的。

首先是样本的处理和裂解步骤，不同样本的裂解条件可能会有所不同，需要根据具体情况进行调整。

其次是磁珠的使用，需要注意磁珠的悬浮情况和使用量，以确保能够有效地结合目标核酸。

在洗涤和洗脱步骤中，也需要注意洗涤缓冲液的温度和使用次数，以及洗脱缓冲液的使用量和温度等细节。

只有严格按照说明书上的要求进行操作，才能够得到高质量的核酸提取物。

三、磁珠法核酸提取试剂盒的个人观点和理解从个人角度来看，磁珠法核酸提取试剂盒无疑是一种高效、便捷的核酸提取工具。

相比传统的有机溶剂提取法和硅基膜法，磁珠法核酸提取试剂盒具有操作简便、提取效率高、纯度好等优点。

在日常的科研工作或临床诊断中，使用磁珠法核酸提取试剂盒能够大大提高工作效率，节约时间和成本。

总结回顾通过本文对磁珠法核酸提取试剂盒的深度评估，相信读者已经对这一产品有了更全面、深刻的理解。

磁珠法基因组DNA 提取试剂盒(细胞)样本前处理：细胞用PBS洗两次后，6000rpm/min 离心3min，弃上清，加入500ul Buffer CGP重悬沉淀，12000rpm/min离心1min，弃上清，取沉淀备用。

2.裂解结合：在细胞沉淀中，加入500μLBuffer CGL，室温放置5-10min，再加入500μl异丙醇和30ul磁珠悬浮液(MB) （使用前充分重悬），颠倒混匀，室温放置5min（期间不时颠倒混匀）；将离心管放置于磁力架上，待磁珠完全吸附后吸弃液体。

3.漂洗:(1)将离心管从磁力架上取下，向离心管中加入500 μl Buffer CGW 0,涡旋振荡5s，重悬磁珠，将离心管放置于磁力架上，待磁珠完全吸附后吸弃液体，重复该步骤1次。

(2)将离心管从磁力架上取下，向离心管中加入500 μl Buffer CGW I,涡旋振荡5s，重悬磁珠,将离心管放置于磁力架上，待磁珠完全吸附后吸弃液体。

重复该步骤一次，待磁珠完全吸附后吸弃液体（液体一定要弃尽，不然残留会影响下游实验），晾干2min。

实验准备56°C加热源磁珠用前一定要充分混匀；使用前需在Buffer CGW I中加入相应体积的无水乙醇。

4.洗脱：将离心管从磁力架上取下加入50-100μl Buffer EB ，涡旋振荡结合移液器吹打，充分吹散磁珠（一定要完全吹散磁珠，若未充分重悬会影响DNA 得率），56°C 放置5min（每隔2min振荡混匀重悬磁珠）,置于磁力架上，将上清DNA转移至一新的离心管中，放入-20 °C保存备用，保质期为2 年。

纯度效果评价通过测定洗脱液中DNA的A260来确定RNA产量，通常情况下A260值在0.1～1.0之间数据比较可信。

如果不在此范围内，请稀释或浓缩样品调整；通过测定洗脱液中RNA的A280和A230来确定DNA纯度，A260/A280比值在1.8-2.0之间，小于1.8表示可能有蛋白质污染。

磁珠的用法1. 磁珠主要用于EMI噪声抑制(可以针对电源，也可以针对信号线)，其直流阻抗(DCR)很小，在高频下却有较高阻抗。

2. 选择磁珠，除了考虑需要选择合适的封装外，主要是关注其：1) 额定电流大小Rated Current (mA)2) 直流阻抗(DCR)DC Resistance (m ohm)3) 阻抗[Z]@100MHz (ohm)/噪声中心频率下的磁珠阻抗(ohm)3. 磁珠阻抗一般指100MHz下的阻抗，比如一个600R的磁珠，表示在100MHz下的阻抗为600欧。

4. 磁珠的参数选择要根据实际情况来进行。

举例说明：假设1) 磁珠左侧输入电源网表: 3.2Vdc + 300mVpp @ 100MHz (后半部分为电源中心频率噪声)2) 磁珠右侧负载要求：Vdc >=3.0VdcVn <= 50mVpp @ 100MHz交流负载>= 50 欧@ 100MHz直流电流<= 300mA那么1) 计算磁珠直流电阻DCR：DCR <= (3.2Vdc-3.1Vdc)/300mA = 0.3 欧2) 计算噪声抑制磁珠阻抗@100MHz >= (300mVpp-50mVpp)/50mVpp*50欧=250欧随意应该选择的磁珠参数为：(1) DCR <= 0.3 欧(2) 100MHz阻抗>= 250 欧(3) 额定电流>= 300 毫安而假设你选取了一个阻抗为50欧的磁珠，那么抑制的效果只有一半，换句话说，在该磁珠右端的输出大概还会有150mVpp的噪声。

另外，从工艺的角度看，上述的(1)和(2)是矛盾的。

所以，选择磁珠之前，你需要先对电路的噪声情况(噪声中心频率、幅度大小、抑制后的大小)和直流情况有一个初步的估计。

然后选择合适的参数。

5. 磁珠名称中的参数含义磁珠一般和电阻一样，用科学技术法表示，比如601表示600欧@100MHz的磁珠。

.血液DNA提取试剂盒（磁珠法）【简介】血液DNA提取试剂盒（磁珠法）适用血液的基因组DNA提取，尤其适用于人类以及哺乳动物的血液基因组DNA提取。

配合核酸自动纯化仪（GNT-SI系列），可实现一键式、自动化操作【参数】名血液基因DN提取试剂GNT-B02货号100T 规格2℃—储存条件30℃360保质期天【组分】100T 规格30ml Buffer LB120mg Buffer LB21ml Magnetic Beads80ml Buffer WB I30ml Buffer WB II11mlElution Buffer【血液DNA提取试剂盒（磁珠法）操作说明】（以实际说明书为准）1、取抗凝全血到离心管中，加入Buffer LB1、Buffer LB2，混合均匀后，将离心管置于水浴锅中，水浴30min2、将离心管取出，加入异丙醇，Magnetic Beads。

颠倒混匀数次（期间应静置数秒后继续颠倒混匀），将离心管置于磁力架，磁分离，吸弃废液3、将离心管从磁力架上取下，加入Buffer WB I。

颠倒混匀数次（期间应静置数秒后继续颠倒混匀），将离心管置于磁力架，磁分离，吸弃废液4、将离心管从磁力架上取下，加入Buffer WB II，颠倒混匀数次（期间应静置数秒后继续颠倒混匀），将离心管置于磁力架，磁分离，吸弃废液5、重复步骤5一次，并将废液完全吸弃干净6、将离心管从磁力架上取下，室温下开盖静置5min7、加入Elution Buffer，磁珠完全混匀后，置于水浴锅中，水浴10min8、将离心管置于磁力架，磁分离，小心吸取上清至新的离心管中，进行下游实验【特点】1、得率高：200ul人类抗凝全血的普遍产量可达3-8ug2、纯度高：OD260/280稳定在1.8左右，OD260/230稳定在1.8以上3、操作简便：手工提取过程无需离心4、自动化：具有成熟的自动化方案，可配合核酸自动纯化仪，实现一键式操作【提取效果】1 / 2.2 / 2。

磁珠的用法磁珠如何使用磁珠（Magnetic beads）是一种常用的实验试剂，主要用于生物学、生物医学、核酸纯化、蛋白质分离、细胞分离等领域。

它具有广泛的应用，能够快速、高效地完成各种实验操作。

1.核酸纯化：磁珠可以与DNA或RNA特异性结合，通过磁力将目标核酸从样品中分离出来。

在核酸纯化过程中，首先需要将磁珠与适当的离心管或微孔板结合，然后将样品加入。

接下来，样品与磁珠发生特异性结合，形成核酸-磁珠复合物。

最后，通过磁力将磁珠集中在离心管或微孔板边缘，将上清液去除，再洗涤、溶解和洗脱核酸。

2.蛋白质纯化：磁珠表面可以修饰特定的亲和基团，能够与具有特异性结合关系的蛋白质结合。

蛋白质纯化过程中，首先将磁珠与合适的亲和基团修饰反应，形成亲和磁珠。

然后将亲和磁珠与待纯化的混合物接触，目标蛋白质与亲和基团发生特异性结合。

最后，通过磁力将磁珠集中在离心管或微孔板边缘，去除上清液，再洗涤、溶解和洗脱目标蛋白质。

3.细胞分离和富集：磁珠可以与细胞标记物结合，用于细胞富集、分离和分析。

在细胞分离过程中，首先将磁珠与合适的细胞标记物结合。

然后将标记的细胞用磁珠分离系统进行分离，通过磁力将目标细胞与磁珠分离出来。

最后，通过去除磁珠，将富集的细胞用于后续实验操作。

4.药物输送：磁珠可以用作药物载体，通过表面修饰的靶向分子与特定的细胞结合，并具有定点释放药物的功能。

在药物输送领域，磁珠被用来增加药物的稳定性和靶向性，提高药物的治疗效果。

总结来说，磁珠的使用涉及到与目标分子（如核酸、蛋白质、细胞等）的特异性结合，通过磁力进行分离、纯化和富集。

在使用磁珠之前，首先需要对磁珠进行适当的修饰，使其具有与目标分子结合的能力。

然后，将样品和磁珠进行接触、结合，通过磁力将目标分子与磁珠分离出来。

最后，对分离、纯化的目标分子进行后续的处理和分析。

需要注意的是，在使用磁珠的过程中，应严格遵守操作规程，确保实验的准确性和安全性。

另外，磁珠的选择应根据实验需求和样品特性进行合理选择，并根据实验要求对磁珠进行适当的处理和修饰。

磁珠纯化原理及操作步骤1 磁珠简介1979 年，John Ugelstad等成功地制备了一种均匀性和粒度适宜的聚苯乙烯微球。

将其磁化并与抗体连接后，即成为一种分离细胞效果极佳的免疫磁珠-Dynabeads。

发展到现在，磁珠并不仅仅表面只包被着抗体，它还有可能表面包被着-COOH、oligo-dT、蛋白A 和G、链霉亲和素等，这为研究人员提供了多样化的选择。

以Agencourt Ampure Beads为例，如下图所示，磁珠由3部分构成，C部分是聚苯乙烯核心，B部分是磁铁微粒，A部分是由羧基组成的多聚物外壳，。

B部分分布的磁铁使磁珠具有独特的超顺磁特性，能保证磁珠只有在磁场中才具有磁性，基于这个特性，磁珠在磁场移走后就很容易重悬且不聚沉。

A部分可特异性分离DNA。

磁珠结构图2 种类目前，国际上使用较为广泛的磁珠有由Backman Coulter提供的Agencourt Ampure Beads以及Invitrogen提供的Dynalbeads。

这些磁珠类型各异，能够特异性分离DNA、RNA、生物素标记的大分子、蛋白质以及细胞。

例如，表面包被有-COOH的磁珠能够特异性分离DNA；表面植有oligo-dT的磁珠可以分离mRNA；表面结合有链霉亲和素的磁珠可以吸附生物素标记的生物大分子；表面包被有抗体的磁珠可以特异性结合抗原性物质。

现我们实验室使用的主要是Agencourt Ampure Beads以分离纯化DNA，以及Invitrogen Dynabeads® M-280 Streptavidin 链霉亲和素磁珠以分离生物素标记的RNA 探针。

3原理Agencourt Ampure Beads的原理是在一定盐（如NaCl、MgCl2、CaCl2、KCl、LiCl 等）和聚乙二醇（PEG-8000）浓度条件下，DNA可非特异性地结合于表面有-COOH末端的磁珠上。

一旦结合，可用70%的乙醇对DNA-磁珠复合物进行彻底清洗，最后用Tris缓冲液将DNA洗脱下来。

`B66食品中布鲁氏杆菌抗原免疫磁珠富集增菌检验方法连云港市产品质量监督检验中心 发布DB32/TDB32/T -2010前言本标准适用于食品中布鲁氏杆菌的检验和鉴定。

本标准编制按GB/T 1《标准化工作导则》和GB/T 20001《标准编写规则》进行。

本标准由连云港市产品质量监督检验中心提出。

本标准由连云港市产品质量监督检验中心负责起草。

本标准主要起草人：本标准于2016年5月首次发布。

食品中布鲁氏杆菌免疫磁珠富集增菌检验方法1 范围本标准规定了食品中布鲁氏杆菌的免疫磁珠富集检验。

本标准适用于牛羊肉和乳制品检测。

本标准检出限为 20CFU/g2 规范性引用文件下列文件中的条款通过本标准的引用而成为本标准的条款。

凡是注日期的引用文件，其随后所有的修改单（不包括勘误的内容）或修订版均不适用于本标准，然而，鼓励根据本标准达成协议的各方研究是否可使用这些文件的最新版本。

凡是不注日期的引用文件，其最新版本适用于本标准。

GB/T 20001.4-2001 标准编写规定实验原理利用免疫学原理，采用布鲁氏杆菌抗血清交联包被在磁珠表面制备成免疫磁珠，当免疫磁珠遇到菌体抗原产生结合，抗原抗体结合完成后再施加外来磁场，使得免疫磁珠贴与试管壁不被洗脱。

然后将磁场消除，磁珠自然落入试管底部，采用非金属接种环或采用加样器将结合菌体的免疫磁珠接种到选择性增菌液中增菌或划线与选择性平板分离培养。

3 设备与材料3.1 低温冷冻高速离心机。

3.2 电动试管振荡器。

3.3 精密恒温水浴箱。

3.4 永磁架3.5 天平（0.1g）3.6 拍打式均质器3.7 冰箱3.8 全支消毒自动加液器（带滤头）3.9 非金属接种环4 试剂及培养基4.1 Dynabeads M-280 磁珠（日本DYNAL社产）或其他牌号的磁珠4.2 牛布鲁氏杆菌抗血清4.3 羊布鲁氏杆菌抗血清4.4 羊抗兔Ig (G)4.5 布鲁氏杆菌选择性琼脂见附录A4.6 布鲁氏杆菌增菌液见附录A4.7 EDC solution 液：500mg EDC in 1ml MES buffer. Use the solution in 2min 4.8 NHS solution 液：250mg NHS in 1ml MES buffer. Use the solution in 2min注：EDC 与 NHS 必须现用现配。

Contents1. Description1.1 Principle of the MACS® Separation1.2 Background information1.3 Applications1.4 Reagent and instrument requirements试剂和仪器的要求2. Protocol2.1 Sample preparation样品制备2.2 Magnetic labeling磁性标记2.3 Magnetic separation磁性分离3. Example of a separation using the CD133 MicroBead Kit4. References1. DescriptionComponents 2 mL CD133 MicroBeads, human:MicroBeads conjugated to monoclonal antihumanCD133 antibodies (isotype: mouse IgG1,clone AC133).2 mL FcR Blocking Reagent, human Specificity CD133 antigen, epitope (CD133/1)1.Capacity For 2亊10⁹total cells, up to 100 separations.Product format CD133 MicroBeads are supplied in buffercontaining stabilizer and 0.05% sodium azide.Storage Store protected from light at 2−8 °C. Do not freeze. The expiration date is indicated on the vial label.1.1 Principle of the MACS® SeparationFirst, the CD133+ cells are magnetically labeled with CD133MicroBeads. Then, the cell suspension is loaded onto a MACS®Column, which is placed in the magnetic field of a MACS Separator.The magnetically labeled CD133+ cells are retained within thecolumn. The unlabeled cells run through; this cell fraction isthus depleted of CD133+ cells. After removing the column fromthe magnetic field, the magnetically retained CD133+ cells can beeluted as the positively selected cell fraction. To increase the purity,the positively selected cell fraction containing the CD133+ cells isseparated over a second column.1.2 Background informationThe CD133 MicroBead Kit is a magnetic labeling system designedfor the positive selection of CD133+ cells. It allows the single-stepisolation of nonhematopoietic and early hematopoietic progenitors and stem cells. The CD133 molecule is a 5-transmembrane cellsurface antigen with a molecular weight of 117 kD.2 The CD133/1(clone AC133) antibody recognizes epitope 1 of the CD133 antigen.1In the hematopoietic system, CD133 expression is restricted to asubset of CD34bright stem and progenitor cells in human fetal liver,bone marrow, cord blood, and peripheral blood.3 Isolated fromhematopoietic sources, CD133+ cells can become adherent and arereported to become CD133–during culture⁴. The CD34+CD133+cell population, which includes CD34+CD38–cells, was shown tobe capable of repopulating NOD/SCID mice.⁵Recently, CD133has also been found to be expressed on circulating endothelialprogenitor cells⁶,⁷and fetal neural stem cells⁸,⁹as well as on othertissue-specific stem cells, such as renal1⁰and prostate11 stem cells.Lately, when isolated from tumor tissue, the CD133+ populationcan be enriched for tumor-initiating cells.11-1⁵CD133 MicroBeadshave been used to isolate adult stem cells from cord blood and asa starting population for reprograming towards iPS cells.1⁶CD133expression has been found on undifferentiated human ES cells.Therefore CD133 MicroBeads could be used for enrichment ordepletion of these cells.1⁷1.3 Applications●Positive selection or depletion of cells expressing human CD133antigen.●Isolation or depletion of CD133+ cells from peripheral bloodmononuclear cells (PBMCs) or single-cell suspensions fromtissue.●CD133+ cells are used in basic stem cell research, stem cellevaluation, stem cell expansion, research in hematologicalmalignancies, stem cell plasticity, and potential cellulartherapies as well as in tissue regeneration and cancer research.1.4 Reagent and instrument requirements●Buffer: Prepare a solution containing phosphate-buffered saline(PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mMEDTA by diluting MACS BSA Stock Solution (# 130‑091-376)1:20 with autoMACS Rinsing Solution (# 130-091-222). Keepbuffer cold (2−8 °C). Degas buffer before use, as air bubblescould block the column.▲Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). BSA can be replaced by other proteins such as human serum albumin, human serum, or fetal bovine serum (FBS). Buffers or media containing Ca2+ or Mg2+ are not recommended for use.●(Optional) Fluorochrome-conjugated antibodies forflow cytometric analysis, e.g., CD133/2 (293C3)-PE(# 130-090-853), CD133/2 (293C3)-APC (# 130-090-854),CD133/2 (293C3)‑Biotin, CD34-FITC (# 130-081-001),CD34‑APC (# 130-090-954), or CD34-PE (# 130-081-002). Formore information about fluorochrome-conjugated antibodiessee .●MACS Columns and MACS Separators: CD133+ cells can beEnriched浓缩by using MS, LS, or XS Columns or depleted withthe use of LD, CS, or D Columns. Cells which strongly expressthe CD133 antigen can also be depleted using MS, LS, or XSColumns. Positive selection正向or depletion can also be performedby using the autoMACS Pro or the autoMACS Separator.▲Note: Column adapters are required to insert certain columns into theV arioMACS™or SuperMACS™Separators. For details see the respective MACS Separator data sheet.●(Optional) Propidium Iodide Solution (# 130-093-233) or7-AAD for flow cytometric exclusion of dead cells.●(Optional) Dead Cell Removal Kit (# 130-090-101) for thedepletion of dead cells.●(Optional) Pre-Separation Filters (# 130-041-407) to removecell clumps团.2. Protocol2.1 Sample preparationWhen working with anticoagulated peripheral blood or buffy coat,peripheral blood mononuclear cells (PBMCs) should be isolated bydensity gradient centrifugation, for example, using Ficoll-Paque™.▲Note: To remove platelets after density gradient separation, resuspend cell pellet in buffer and centrifuge at 200×g for 10−15 minutes at 20 °C. Carefully aspirate supernatant. Repeat washing step.When working with tissues or lysed blood, prepare a single-cellsuspension using standard methods.For details see the protocols section at /protocols.For preparation of cord blood cells, bone marrow cells, or cellsfrom leukapheresis material, please refer to the sample preparationprotocols at /protocols.▲Dead cells may bind non-specifically to MACS MicroBeads.To remove dead cells, we recommend using density gradientcentrifugation or the Dead Cell Removal Kit (# 130-090-101).2.2 Magnetic labeling▲Work fast, keep cells cold, and use pre-cooled solutions. This willprevent capping of antibodies on the cell surface and non-specificcell labeling.▲V olumes for magnetic labeling given below are for up to10⁸total cells. When working with fewer than 10⁸cells, use the same volumes as indicated. When working with higher cell numbers,scale up all reagent volumes and total volumes accordingly (e.g.for 2亊10⁸total cells, use twice the volume of all indicated reagent volumes and total volumes).▲For optimal performance it is important to obtain a single‑cell suspension before magnetic labeling. Pass cells through 30 μm nylonmesh (Pre-Separation Filters, # 130-041-407) to remove cell clumpswhich may clog the column. Moisten filter with buffer before use.▲The recommended incubation temperature is 2–8 °C. Workingon ice may require increased incubation times. Higher temperaturesand/or longer incubation times may lead to non-specific celllabeling.1. Determine确定cell number.2. Centrifuge cell suspension细胞悬液at 300×g for 10 minutes. Aspirate supernatant completely.吸取上清3. Resuspend cell pellet in 300 μL of buffer per 10⁸total cells.4. Add 100 μL of FcR Blocking Reagent per 10⁸total cells.5. Add 100 μL of CD133 MicroBeads per 10⁸total cells.6. Mix well and incubate for 30 minutes in the refrigerator(2−8 °C).7. (Optional) Add staining antibodies, e.g., 50 μL of CD133/2(293C3)-PE (# 130-090-853), and incubate for 5 minutes in thedark in the refrigerator (2−8 °C).8. Wash cells by adding 1−2 mL of buffer per 10⁸cells andcentrifuge at 300×g for 10 minutes. Aspirate supernatantcompletely.9. Resuspend up to 10⁸cells in 500 μL of buffer.▲Note: For higher cell numbers, scale up buffer volume accordingly.▲Note: For depletion with LD Columns, resuspend up to 1.25亊10⁸cells in 500 μL of buffer.10. Proceed to magnetic separation (2.3).2.3 Magnetic separation▲Choose an appropriate MACS Column and MACS Separatoraccording to the number of total cells and the number of CD133+cells. For details see table in section 1.4.▲Always wait until the column reservoir is empty before proceedingto the next step.Magnetic separation with MS or LS Columns▲To achieve highest purities, perform two consecutive columnruns.1. Place column in the magnetic field of a suitable MACS Separator.For details see the respective MACS Column data sheet.2. Prepare column by rinsing with the appropriate amount ofbuffer:MS: 500 μL LS: 3 mL3. Apply cell suspension onto the column. Collect flow-throughcontaining unlabeled cells.4. Wash column with the appropriate amount of buffer. Collectunlabeled cells that pass through and combine with the effluentfrom step 3.MS: 3×500 μL LS: 3×3 mL▲Note: Perform washing steps by adding buffer aliquots only when the column reservoir is empty.5. Remove column from the separator and place it on a suitablecollection tube.▲Note: To perform a second column run, you may elute the cells directly from the first onto the second, equilibrated column instead of a collection tube.6. Pipette the appropriate amount of buffer onto the column.Immediately flush out the magnetically labeled cells by firmlypushing the plunger into the column.MS: 1 mL LS: 5 mL7. To increase purity of CD133+ cells, enrich the eluted fractionover a second MS or LS Column. Repeat the magneticseparation procedure as described in steps 1 to 6 by using anew column.Magnetic separation with XS ColumnsFor instructions on the column assembly and the separation refer tothe XS Column data sheet.Depletion with LD Columns1. Place LD Column in the magnetic field of a suitable MACSSeparator. For details see LD Column data sheet.2. Prepare column by rinsing with 2 mL of buffer.3. Apply cell suspension onto the column.4. Collect unlabeled cells that pass through and washcolumn with 2×1 mL of buffer. Collect total effluent;this is the unlabeled cell fraction. Perform washingsteps by adding buffer two times. Only add new buffer whenthe column reservoir is empty.Depletion with CS Columns1. Assemble CS Column and place it in the magnetic field of asuitable MACS Separator. For details see CS Column datasheet.2. Prepare column by filling and rinsing with 60 mL of buffer.Attach a 22G flow resistor to the 3-way stopcock of theassembled column. For details see CS Column data sheet.3. Apply cell suspension onto the column.4. Collect unlabeled cells that pass through and wash columnwith 30 mL buffer from the top. Collect total effluent; this isthe unlabeled cell fraction.Depletion with D ColumnsFor instructions on column assembly and separation refer to theD Column data sheet.Magnetic separation with the autoMACS® Pro Separator or theautoMACS® Separator▲Refer to the respective user manual for instructions on how to use the autoMACS Pro Separator or the autoMACS Separator.▲Buffers used for operating the autoMACS Pro Separator or the autoMACS Separator should have a temperature of ≥10 °C.▲Program choice depends on the isolation strategy, the strengthof magnetic labeling, and the frequency of magnetically labeled cells. For details refer to the section describing the cell separation programs in the respective user manual.Magnetic separation with the autoMACS® Pro Separator1. Prepare and prime the instrument.2. Apply tube containing the sample and provide tubes for collecting the labeled and unlabeled cell fractions. Placesample tube in row A of the tube rack and the fractioncollection tubes in rows B and C.3. For a standard separation choose one of the following programs:Positive selection from peripheral blood, bone marrow, or leukapheresis: “Posseld”Collect positive fraction in row C of the tube rack.Positive selection from cord blood: “Posseld2”Collect positive fraction in row C of the tube rack.Depletion: “Depletes”Collect negative fraction in row B of the tube rack.Magnetic separation with the autoMACS® Separator1. Prepare and prime the instrument.2. Apply tube containing the sample and provide tubes for collecting the labeled and unlabeled cell fractions. Placesample tube at the uptake port and the fraction collectiontubes at port neg1 and port pos 2.3. For a standard separation choose one of the following programs:Positive selection from peripheral blood, bone marrow, or leukapheresis: “Posseld”Collect positive fraction from outlet port pos 2.Positive selection from cord blood: “Posseld2”Collect positive fraction from outlet port pos 2.Depletion: “Depletes”Collect negative fraction from outlet port neg1.3. Example of a separation using the CD133MicroBead KitCD133+ hematopoietic stem and progenitor cells were isolated from non-mobilized human PBMCs using the CD133 MicroBead Kit, MS Columns, and a MiniMACS™Separator. Cells were fluorescently stained with CD34-FITC (# 130-081-001) and CD133/2 (293C3)-PE (# 130-090-853) and analyzed by flow cytometry. Cell debris and dead cells werde excluded from the analysis based on scatter signals and propidium iodide fluorescence.All protocols and data sheets are available at .WarningsReagents contain sodium azide. Under acidic conditions sodium azide yields hydrazoic acid, which is extremely toxic. Azide compounds should be diluted with running water before discarding. These precautions are recommended to avoid deposits in plumbing where explosive conditions may develop.WarrantyThe products sold hereunder are warranted only to be free from defects in workmanship and material at the time of delivery to the customer. Miltenyi Biotec GmbHmakes no warranty or representation, either expressed or implied, with respect tothe fitness of a product for a particular purpose. There are no warranties, expressedor implied, which extend beyond the technical specifications of the products.Miltenyi Biotec GmbH’s liability is limited to either replacement of the products or refund of the purchase price. Miltenyi Biotec GmbH is not liable for any property damage, personal injury or economic loss caused by the product.autoMACS and MACS are registered trademarks and MidiMACS, MiniMACS, OctoMACS, QuadroMACS, SuperMACS, and V arioMACS are trademarks of Miltenyi Biotec GmbH.Ficoll-Paque is a trademark of GE Healthcare companies.Copyright . 2009 Miltenyi Biotec GmbH. All rights reserved.。