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View additional product information for CELLection™ Epithelial Enrich Dynabeads™ - FAQs (16203)
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不可以，当细胞与微珠结合时，所得的数据并不理想。您需要通过Dynabeads 试验去除细胞上结合的微珠，从而进行下游分析。适于这一类应用的检测体系包括Dynabeads FlowComp 检测和DETACHaBEAD检测。
无珠细胞能够通过使用DETACHaBEAD技术的Dynabeads试剂盒分离得到。这些包括了Dynabeads FlowComp试剂盒和Dynabeads CELLection细胞分离试剂盒。同时，任何阴性分离试剂盒都会产生无珠细胞。
人类细胞
Dynabeads FlowComp 人 CD4试剂盒（T 细胞）（货号11361D）
Dynabeads FlowComp人CD8试剂盒（T 细胞）（货号11362D）
Dynabeads FlowComp人CD3试剂盒（T 细胞）（货号11365D）
Dynabeads FlowComp人CD14试剂盒（T 细胞）（货号11367D）
Dynabeads CD34阳性分离试剂盒（造血祖细胞）（货号11301D）
Dynabeads CD4阳性分离试剂盒（T 细胞）（货号11331D）
Dynabeads CD8阳性分离试剂盒（T 细胞）（货号11333D）
Dynabeads 标准CD4+/CD25+ T 细胞试剂盒（货号11363D）
DETACHaBEAD CD19试剂盒（货号12506D）
CELLection 上皮细胞富集的 Dynabeads（货号16203）
Dynabeads人树状细胞（DC）富集试剂盒（货号11308D）
小鼠细胞
Dynabeads FlowComp小鼠CD4试剂盒（T 细胞）（货号11461D）
Dynabeads FlowComp小鼠CD8试剂盒（T 细胞）（货号11462D）
Dynabeads FlowComp小鼠CD4+CD25+调节性T细胞试剂盒（货号11463D）
Dynabeads FlowComp小鼠Pan T（CD90.2）（货号11465D）
Dynabeads小鼠Pan T（CD90.2）（货号11443D）
DETACHaBEAD小鼠CD4试剂盒（T 细胞）（货号12406D）
这一换算需基于具体的磁珠尺寸，通常情况下，对于M-450磁珠而言，1 mg/mL磁珠大约相当于1.3 X 10E7个磁珠/mL；对于M-280磁珠来说，1 mg/mL磁珠大约相当于6.7 X 10E7个磁珠/mL。
在培养中细胞是否会内吞Dynabeads磁珠要视具体细胞类型而定。由于磁珠尺寸的关系（通常直径为4.5 μM），Dynabeads磁珠将不会通过内吞途径（例如通过网格蛋白有被小窝）发生细胞内化。网格蛋白有被小窝在尺寸上一般不会大于500 nm，这对于磁珠的内吞来说太小了。不过，如果细胞具有吞噬活性（如单核细胞/巨噬细胞），Dynabeads磁珠还是可能在这些特定类型的细胞中被吞噬进入吞噬溶酶体的。所以您问题的答案可能为是或否——视具体的细胞类型而定。
我们提供了多种不含释放机制的Dynabeads磁珠，这些产品可用于细胞阳性分离（捕获靶标细胞）或去除操作（将靶标细胞从样本中去除）：
•适用于细胞去除操作的Dynabeads磁珠：通过Dynabeads磁珠进行的细胞清除是一种十分快速、高效和方便的技术。选用预先包被的Dynabeads磁珠，或使用您的自备靶标抗体来包被我们的二抗包被磁珠，将其加入样本（如全血、PBMC、白细胞层，组织消化产物），混匀并孵育20分钟，再置于磁力架2分钟，您就完成了细胞去除操作。
•适用于阳性分离操作的Dynabeads磁珠，可匹配分子水平的下游分析应用：不含磁珠释放机制的靶细胞阳性分离可用于DNA、RNA或蛋白分析等分子水平的下游研究。在这些应用中，当磁珠连着细胞时，可对分离的细胞进行裂解，当细胞裂解后，磁珠可被移除。如果体系中存在磁珠不是问题，您也可在磁珠存在的条件下培养细胞。在大多数情况下，2-3天后表面的抗原会发生内化，磁珠也会随之脱落——因为这些磁珠相对体积过大，而无法通过细胞内吞途径来实现内化。
在通常情况下，Dynabeads磁珠的尺寸很大，不会发生细胞内吞的现象。网格蛋白有被小窝通常不会大于500 nm，这对Dynabeads磁珠的内吞作用来说太小了。不过，对于像单核细胞/巨噬细胞等具有吞噬活性的靶细胞而言，Dynabeads磁珠还可能由吞噬作用介导进入细胞内部。
CELLection试剂盒中包含了经DNA接头包被（可通过链霉亲和素或抗体用于细胞分离）的Dynabeads磁珠，和一款用于释放磁珠的DNA酶。在使用磁珠（直接或间接地）捕获靶标细胞后，可通过DNA酶的处理来剪切DNA接头，以释放细胞。其结果是，靶细胞从Dynabeads磁珠上解离下来，但仍与捕获抗体相结合。
我们提供了三种Dynabeads CELLection细胞分离试剂盒：
•CELLection上皮细胞富集试剂盒（货号16203）中包含了与anti-EpCAM单抗相偶联的Dynabeads磁珠。
•CELLection生物素结合剂试剂盒（货号11533D）适合应用您自备的生物素化抗体来对靶标细胞进行阳性分离
•CELLection泛小鼠IgG试剂盒（货号11531D）适合应用您自备的小鼠IgG来分离靶标细胞
CELLection上皮细胞富集试剂盒所提供的磁珠上包被有Ber-EP4，该分子能够以高亲和力与上皮细胞标志物EpCAM（上皮细胞粘附分子）相结合。上皮细胞富集产品无法阳性分离循环肿瘤细胞（circulating tumour cells），但为科研人员提供了一种能够直接应用于全血、骨髓或MNC样本的高灵敏度富集技术。本产品基于健康（亦或无循环肿瘤细胞，circulating tumour cells）个体的血流中不存在上皮细胞的理论。因此，从血样中分离出上皮细胞是存在循环肿瘤细胞（circulating tumour cells）的一个重要提示，但还需凭借下游检测来进行确认。除上皮细胞外，一些白细胞也会被捕获或由于非特异性结合而被分离出来。不过这些白细胞的比例显著低于上皮细胞，该领域的研究者可通过肉眼简单地从上皮细胞富集磁珠共分离出的细胞中鉴定或分辨出上皮（肿瘤）细胞。5 mL血样中可能有1万乃至更多数量的白细胞伴随着上皮细胞被共同分离出来。所分离出的细胞应随即进行下游研究来（1）验证上皮细胞属性（如通过细胞角蛋白等进行免疫染色），之后（2）进一步针对所富集细胞进行肿瘤细胞的确认和鉴定。
EpCAM抗原是一种上皮细胞粘附分子，在绝大多数上皮来源的细胞，以及上皮来源的肿瘤细胞上高表达。因此，在正常情况下的循环血液中不含上皮细胞的前提下，从血液、单核细胞或骨髓样本中分离肿瘤细胞时，珠子上的Ber-EP4(抗epCAM)抗体将识别正常和癌变的上皮细胞。
有三种方法可去除所分离细胞上结合的Dynabeads磁珠。
•DETACHaBEAD 试剂盒（阳性分离）——解离试剂为抗Fab的多抗试剂，将磁珠结合的抗体从细胞上竞争性结合下来，从而帮助用户获得不含抗体和磁珠的细胞。提供适用于人CD4+和CD8+ T细胞，CD19+ B细胞和CD34+造血干细胞的试剂盒。
•CELLection试剂盒——解离试剂是一种DNA酶，能够消化抗体与磁珠之间的DNA接头，从而帮助用户最终获得不含磁珠的细胞。提供适用于人体EpCAM（Ber-EP4）上皮细胞和链霉亲和素（能够结合任一种生物素标记的抗体）的试剂盒。
•FlowComp试剂盒——解离试剂为生物素，能够竞争性结合与磁珠相偶联的des-生物素化抗体。提供适用于人类和小鼠总T细胞，CD4+和CD8+的T细胞亚群，以及人单核细胞的试剂盒。
在细胞阳性分离操作中，我们推荐您在每mL样品中使用1–2 x 10E7总PBMC/mL，和1 x 10E7个Dynabeads磁珠/mL样品。另一种确定所需磁珠用量的方法是，假设为每个靶标细胞提供4个左右的磁珠。如果您希望去除样本中某一类靶标细胞，我们推荐您使用与上述情况相同的细胞浓度（1–2 x 10E7 总PBMC/mL），但至少将Dynabeads磁珠的用量翻倍至2 x 10E7个Dynabeads磁珠/mL样品。或者，假设为每个需去除的靶标细胞提供8-10个左右的磁珠。
注意：大多数用于细胞分离的Dynabeads磁珠是以约4 x 10E8个磁珠/mL的浓度来提供的，因此1 x 10E7个微珠的体积仅有25 μL（请检查每一产品的特定产品手册或标签来进行确认）。
阳性分离法的主要优势为高纯度和高得率。此外，阳性分离法一般不需要初始的样本制备步骤，因为细胞可直接从全血、骨髓或组织消化产物等任何样品中直接被分离出来。如果使用一抗包被磁珠且无需释放磁珠，这一分离步骤就非常简单迅速；举例来说，您可在25分钟之内从全血样本中直接分离出细胞，同时可获得非常高的细胞活力。任何阳性分离法的普遍缺点为对细胞的潜在激活效应，这是由靶标细胞上靶抗原表位的功能来决定的。请注意Dynabeads磁珠不会直接导致细胞激活效应；导致此效应发生的是抗体-细胞抗原表位之间的相互作用。当使用带有磁珠释放机制的阳性分离法时，必然会损失一定的细胞得率，因为细胞与磁珠之间的所有连接不可能被完全打断。
阳性分离是一种从样本中分离单一特异性靶细胞群体的方法。通过使用靶标特异性抗体包被的Dynabeads磁珠，磁珠只会与表面携带匹配抗原的靶细胞进行结合。用户可基于下游应用，在基于Dynabeads磁珠的两种不同阳性分离法中作出选择。由于磁珠自身体积就很大（直径在1 µM至4.5 µM之间），因此我们不推荐您在任何类型的流式细胞仪中直接使用磁珠结合的细胞。因此，如果您需要开展下游的细胞学分析，并希望使用流式细胞仪，我们推荐您使用我们能够释放磁珠的阳性分离试剂盒。另一方面，如果您希望将分离出的细胞应用于下游的分子研究，您无需从细胞上释放磁珠，可使用不含任何释放机制的直接交联的磁珠。
不可以，PBS中不能含有Ca2+和Mg2+，因为二价阳离子会导致补体激活和细胞聚集。样品中发生细胞聚集会同时严重降低细胞得率和分离纯度。分离缓冲液中必须加入EDTA，以减少补体激活和细胞聚集效应。另一种选择是以0.6%的柠檬酸钠代替EDTA。
当细胞纯度是最重要指标，而无需顾及靶细胞的激活问题，或下游应用为分离RNA或gDNA时，推荐使用阳性分离法。当最重要的指标是尽可能减少对所分离细胞的干扰，以及下游应用包括细胞培养，研究细胞功能、形态和流式细胞术时，则推荐阴性分离法（请注意阳性分离细胞也可用于包括细胞培养、流式细胞术或细胞功能学研究等下游应用，不过由于阳性分离法中需要从细胞上释放磁珠，用户可使用例如FlowComp产品或含有DETACHaBEAD产品的阳性分离试剂盒完成这一操作）。
在37°C水浴中对细胞冻存管中的细胞进行解冻，直至剩余一个小冰晶时取出。解冻后立即将细胞轻柔地转入10-15 mL的新离心管中，向细胞中逐滴加入10 mL 20% FCS/人血清，并温和的吹打。避免气泡产生并尽快完成操作。以200 x g离心细胞8分钟。弃去上清。在适当的缓冲液/培养基中重悬细胞。
通常情况下，冻存培养基（10% DMSO和90% FCS）或Gibco Recovery细胞冻存培养基（货号12648-010）的使用效果都很好。冻存过程中总有一些细胞会发生死亡。此外，冻存和复苏过程可能会造成某些细胞的裂解。请按以下步骤使用Gibco Recovery培养基冻存哺乳动物细胞：
1.化冻Recovery细胞冻存培养基，彻底混匀后在2–8°C放置，直至使用。
2.对于悬浮细胞从步骤3开始操作。对于贴壁细胞而言，用户应使用Gibco TrypLE试剂等适当的解离试剂，将细胞从其生长基质上轻柔地解离下来。使用该细胞的完全培养基重悬细胞。
3.将细胞悬液移至15-mL的无菌离心管中。
4.使用Invitrogen Countess自动细胞计数仪（也可使用类似的自动或手动方法）确定活细胞的密度和百分比，并计算所需Recovery细胞冻存培养基的体积，使最终细胞密度达到1 × 10E6至1 × 10E7个细胞/mL。
5.100-200 × g离心细胞悬液5-10分钟。在无菌条件下倒出上清液，但不要扰动细胞沉淀。注意：离心速度和时间可基于具体细胞类型来进行调整。
6.使用（2–8°C）冷却的Recovery细胞冻存培养基，以基于具体的细胞类型而推荐的活细胞密度（通常为1 × 10E6个细胞/mL或更高）重悬细胞沉淀。
7.按照生产商的技术说明（即在2 mL冻存管中加入1.5 mL悬液）将细胞悬液分装于冻存管中（应不时地轻柔混匀，以维持细胞悬液均匀）。
8.使用自动或手动控制变温速率的冷冻装置，按照标准步骤执行细胞冻存操作（约每分钟降低1°C左右）。
9.将冻存细胞移入液氮（气相）中，推荐保存于–200°C至–125°C。
加入Dynabeads磁珠之前需对骨髓进行洗涤和稀释，以降低样本粘性。在制备骨髓细胞的过程中，推荐在使用Dynabeads磁珠分离细胞之前，对样本进行洗涤和DNA酶处理：
1.将2 mL（10E7-10E8个细胞）骨髓样本与2 ml PBS w/ 0.1% BSA + 0.6%柠檬酸钠溶液进行混合。
2.在18-25°C条件下以600 g离心8分钟。
3.弃去上清，再使用5 mL 含0.1% BSA + 1mM CaCl2 + 0.5 mM MgCl2的PBS进行重悬。
4.加入600个Kunitz单位的DNase I（120个Kunitz单位DNase I/毫升）。
5.在18-25°C条件下孵育细胞30分钟，期间不断地进行轻柔的倾斜和旋转。
6.在18-25°C条件下以600 g对细胞悬液离心8分钟。
7.弃去上清，在5 mL含0.1% BSA的 PBS 中重悬细胞。
8.在18-25°C条件下以600 g对细胞悬液离心8分钟。
9.弃去上清，使用RPMI 1640 / 1% FCS将细胞重悬至1 x 10E8个细胞/毫升的浓度。
使用酶解消化和机械破碎等标准组织处理方法来制备单细胞悬液。通过细胞筛或30 µm滤网对消化后的细胞悬液进行过滤，以减少大块的聚集物。组织破碎通常会导致部分细胞死亡并释放出DNA。游离DNA会降低细胞捕获效率，回收率和纯度。DNase I处理的方法是：在18–25°C条件下将细胞悬液与含0.1% BSA + 1 mM CaCl2 + 0.5 mM MgCl2的PBS溶液以及120 Kunitz单位DNase I/mL共同孵育30分钟（对于CELLection产品，需要在加入磁珠前对细胞进行洗涤以去除DNA酶）。
MNC也称为外周血单核细胞（PBMC），是从全血、白细胞层、骨髓或脐带血中通过密度梯度离心分离出来的。下面介绍了用于阳性分离或去除法实验方案的标准MNC制备实验方案：
1.收集含抗凝剂（EDTA，ACD，肝素）的血样。分别按照外周血1+1，白细胞层1+2，骨髓1+1和脐带血1+3的比例，使用PBS （含0.1% BSA+ 0.6%柠檬酸钠或2 mM EDT）进行稀释。
2.取一个50 mL离心管，将35 mL稀释后的样本加在15 mL梯度介质（如Ficoll或Lymphoprep溶液）上层。
3.在18–20°C条件下以400 x g离心30-40分钟。如果血液保存时间超过两小时，则再增加10分钟离心时间。
4.收集中间层中的MNC，并将这些细胞移至50 mL离心管中。
5.使用含0.1% BSA的PBS 洗涤MNC三次，其间通过2–8°C300 x g离心8分钟的操作来沉淀细胞。
6.使用含0.1% BSA的PBS溶液将细胞重悬至1 x 10E7个细胞/毫升，并冷却至2–8°C。
请注意：MNC中包含T细胞（50%），B细胞（5-10%），NK细胞（5-10%）和单核细胞（30%），极少量的血小板且不含粒细胞。
如后续需要使用非接触式/阴性分离试剂盒，则推荐使用下列方案来制备低血小板含量和最高纯度的MNC：
可使用全血/白细胞层和骨髓样本作为起始样本。
1.在18–25°C条件下使用PBS （含0.1% BSA+ 0.6%柠檬酸钠或2 mM EDTA），将10-18 mL血液/白细胞层样品稀释至35 mL。
2.将稀释后的血液/白细胞层样本加至15 mL梯度介质（如Lymphoprep或Ficoll溶液）上方。
3.在20°C条件下以160 x g离心20分钟。离心结束后逐渐减速而不要骤然停止。
4.去除20 mL上清液，以减少血小板组份。
5.在20°C条件下以350 x g离心20分钟。离心结束后逐渐减速而不要骤然停止。
6.从血浆/Lymphoprep溶液的中间层收集MNC，并将这些细胞移至50 mL离心管中。
7.使用含0.1% BSA的PBS洗涤MNC一次，通过2–8°C下400 x g离心8分钟来沉淀细胞。
8.使用含0.1% BSA的PBS洗涤MNC两次，通过2–8°C下225 x g的离心8分钟来沉淀细胞。使用含0.1% BSA的PBS，以1 x 10E8个MNC/毫升的密度重悬MNC。
白细胞层，即白细胞浓缩层，是对抗凝血样进行离心（不使用例如Ficoll溶液等密度梯度试剂）后获得的中间层组份，位于血浆下方，红细胞上方。白细胞层同时含有白细胞和血小板，因此可作为这些细胞材料的来源。
通常情况下，1毫升成年人血中包含：
~5 x 10E9 个红细胞
~7 x 10E6 个白细胞
~3 x 10E8 个血小板
在7 x 10E6个白细胞组份中，包含：
4 x 10E5个单核细胞
1 x 10E5个NK细胞
淋巴细胞：
2 x 10E5个B细胞
1 x 10E6个T细胞（约70%为CD4+ T细胞，30%为CD8+ T细胞）
粒细胞：
5 x 10E6个嗜中性白细胞
2 x 10E5个嗜酸性细胞
4 x 10E4个嗜碱性细胞
这要视您的具体应用而定。一般来说，4.5微米的磁珠最适合细胞分离和激活/扩增。这些较大的磁珠具有更高的磁流动性，而且尺寸上与哺乳动物细胞近似，因而不大可能被细胞吞噬。较小的1微米磁珠和2.8微米磁珠通常用于分离核酸或蛋白，或用于免疫沉淀实验。阴性分离细胞试剂盒中通常使用1微米的磁珠，因为它们具有更高的每毫升结合能力和更快的结合动力学。在阴性分选过程中，细胞对磁珠的吞噬不会成为问题，因为用户只需对剩余的细胞群体进行观察。用二抗，蛋白A或蛋白G，或链霉亲和素包被的2.8微米Dynabeads磁珠，配合使用自选的一抗，靶向结合特异性的细胞表面抗原，也可应用于阳性细胞筛选。
我们提供三种不同大小的Dynabeads磁珠：1微米的磁珠（在产品名称中搜索Invitrogen MyOne磁珠），2.8微米的磁珠和4.5微米的磁珠。通常情况下，每毫升磁珠的结合能力和结合动力学参数随着磁珠尺寸的减小而增大。
Dynabeads 磁珠具有超强的顺磁性，这意味着它们只在磁力架作用的条件下表现出磁性。一旦移除磁力架，磁珠就可像液体一样操作，也可方便地分装于样品管中。在细胞分离的相关应用中，这些特性具有显著的优势——能够实现轻柔的操作，以减少细胞承受的压力。第二，这些磁珠的形状和大小均一，并具有快速的液相反应动力学性质。磁珠光滑的表面能够有助于减少非特异性结合。这些性质能够降低体系的变异度，从而帮助您在纯化和分析过程中获得更为可靠和重复性良好的实验结果——无论您需要观察细胞还是其他靶分子（RNA/DNA/蛋白质/蛋白复合物/细胞器/外泌小体等等。）
这里列举了一些建议：
•请确保RPMI+1% FBS的pH值不要太高：DNA酶I在pH 7.0–7.4之间的效率最高（RPMI暴露于大气环境中会导致pH值增加，pH指示剂会变紫）。
•请勿在DNA酶I的处理过程中使用RPMI+10% FCS。应用10%的FBS会比1%的FBS获得的细胞得率更低（可能由于一些批次的FBS中含有的DNA酶I抑制因子）。
•请确保缓冲液中含有DNA酶活性所需的Mg2+/Ca2+。
•DNA酶I必须温和处理。剧烈搅拌DNA酶溶液会降低酶活性。一定不要对DNA酶溶液进行涡旋操作。
•当回收较低数量的细胞时，我们推荐您使用RPMI+1% FBS培养基预先包被管体，以减少细胞的损失（细胞很粘，容易在分选过程中贴附于管壁上）。
•DNA酶I在20°C条件下具有良好活性，不过，我们推荐用户在DNA酶I的处理步骤之前将RPMI+1% FBS预热至37°C，因为不同实验室的室温并不相同。
•当细胞与DNA酶释放缓冲液孵育后，在使用磁力架分离之前对磁珠-细胞混合物进行彻底的吹打非常重要，这样可提供机械力打断DNA连接。未仔细吹打细胞将会降低细胞的得率。
这里列举了一些建议：
•由于DSB-X 生物素-链霉亲和素之间的结合随着时间延长而逐渐变强；因此不要将释放步骤的时间延长至实验方案中规定的时间以上。在某些案例中，更短的孵育时间会获得更高的得率。
•在与解离试剂孵育后，我们推荐您在将样本放置到磁力架上之前，使用1 mL枪头吹打样品10次以上，以充分重悬样本。
这里列举了一些建议：
•样本制备过程非常重要。所有的分离操作都必须在单细胞悬液中进行。对于直接从全血样本中分离某一类型细胞（如单核细胞）的操作而言，我们推荐您在分离前对血样进行洗涤，以去除其中的干扰因素。
•对于指定了“混匀”操作的所有孵育过程而言，必须使用一款合适的混合器。可使用能够同时提供倾斜和旋转功能或倒转混合功能的混合器（如HulaMixer样品混合器（目录编号15920D））。
•如使用FlowComp试剂盒，在加磁珠前先在细胞中加入抗体（间接法），则应在加磁珠前通过洗涤去除过量的抗体。
•在与解离试剂孵育后，我们推荐您在将样本放置到磁力架上之前，使用1 mL枪头吹打样品10次以上，以充分重悬样本。
•用户须在细胞分离过程中使用推荐的分离缓冲液。PBS中不可含有Ca2+和Mg2+，因为二价阳离子会导致补体激活和细胞聚集。
•样品中发生细胞聚集会同时严重降低细胞分离的得率和纯度。
CELLection Epithelial Enrich reagent is bound to the beads via a cleavable DNA linker. This provides you with the option to release the isolated cells from the beads using the supplied release buffer, which returns cells with no beads attached for further cell culture or functional assays. CELLection Epithelial Enrich reagent is recommended for processing a total of 2 x 10E9 cells. The CELLection Epithelial Enrich beads are coated with a mouse anti-BerEP4 monoclonal antibody to the human epithelial cell adhesion molecule (EpCAM).
By making a single-cell suspension of the tissue, it should be possible to isolate the cells using CELLection Epithelial Enrich reagent (for cellular applications) or Dynabeads Epithelial Enrich reagent (for molecular applications). The difference between the CELLection Epithelial Enrich product and the Dynabeads Epithelial Enrich product is that the primary antibody on the CELLection product is coupled to the beads via a DNA linker, providing this bead with a release mechanism via the DNase containing release buffer supplied with the kit. This product is intended for isolation of cells that need to be bead free for downstream studies, while the Dynabeads Epithelial Enrich product is intended for molecular applications (e.g. DNA or mRNA isolation). However, since the antibody coated onto these beads recognizes EpCAM, this epitope needs to be expressed on the cells.
The CELLection Epithelial Enrich product is intended for enrichment of tumor cells rather than isolation of pure cells. This is because the number of target cells can often be as low as 1 tumor cell in one million cells, making it very difficult to get rid of all the contaminating cells even after rigorous washing. This is why we also recommend that molecular analysis or visual morphological verification is necessary to verify that the isolated cells are indeed tumor cells.
To ensure the most efficient cell capture:
Always titrate the antibody concentration used for coating the CELLection Dynabeads magnetic beads and test the direct and the indirect technique. We suggest using 1 x 10e7 beads per mL sample (25 µL) for efficient positive isolation. Incubate at 2 - 8 degrees C with tilting and rotation. When using whole blood samples, wash whole blood as described before use.
To ensure the most efficient cell release:
Never vortex DNase when enzyme is in solution, as this may destroy enzymatic activity. For cell release, use fresh RPMI plus 1% FBS prewarmed to 37 degrees C to ensure the DNase is active. The pH of the RPMI should be 7.0 to 7.5. Higher pH will inhibit DNase activity. Optimal DNase activity requires divalent ions (Mg2+, Ca2+, Mn2+). Generally, no increased release is observed when additional divalent ions are added to the DNase Releasing Buffer, but for this option, use one part of 10x Tris to 9 parts RPMI. (10x Tris = 400 mM Tris-HCl, 100 mM MgSO4, 10 mM CaCl2, 10% fetal calf serum). After cells are incubated with DNase Releasing Buffer, it is essential that the bead-cell complexes are vigorously pipetted before magnetic separation to mechanically disrupt the DNA linker. Failure to pipette the cells will affect cell yield.
Both bead-to-target cell ratio and the concentration of beads in the bead/cell mixture are important and should be considered. For example, when using the Dynabeads magnetic beads M-450 CD4 positive isolation or depletion kit, a 4:1 bead-to-target cell ratio should be maintained. To capture 95% of target cells for molecular applications, the bead concentration must always be 1 x 10e7 beads per milliliter of sample. To deplete 99% CD4 cells from the starting sample, the bead concentration must always be 2 x 10e7 beads per milliliter of sample. Please consult the package insert for recommended bead concentrations of each product.
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No, the data are not ideal when cells have beads attached to them. You need to use one of the Dynabeads Assays that allow you to remove the beads from your cells for downstream analysis. These include the Dynabeads FlowComp assays and DETACHaBEAD assays.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Bead-free cells can be isolated using Dynabeads kits that include DETACHaBEAD technology. These include the Dynabeads FlowComp kits and Dynabeads CELLection Cell Isolation Kits. Also, any of the negative isolation kits will generate cells that are bead-free.
Human Cells
Dynabeads FlowComp Human CD4 Kit (T cells) (Cat. No. 11361D)
Dynabeads FlowComp Human CD8 Kit (T cells) (Cat. No. 11362D)Dynabeads FlowComp Human CD3 Kit (T cells) (Cat. No. 11365D)
Dynabeads FlowComp Human CD14 Kit (monocytes) (Cat. No. 11367D)
Dynabeads CD34 Positive Isolation Kit (hematopoietic progenitor cells) (Cat. No. 11301D)
Dynabeads CD4 Positive Isolation Kit (T cells) (Cat. No. 11331D)
Dynabeads CD8 Positive Isolation Kit (T cells) (Cat. No. 11333D)
Dynabeads Regulatory CD4+/CD25+ T Cell Kit (Cat. No. 11363D)
DETACHaBEAD CD19 Kit (Cat. No. 12506D)
CELLection Epithelial Enrich Dynabeads (Cat. No. 16203)
Dynabeads Human Dentritic Cells (DC) Enrichment Kit (Cat. No. 11308D)
Mouse Cells
Dynabeads FlowComp Mouse CD4 Kit (T cells) (Cat. No. 11461D)
Dynabeads FlowComp Mouse CD8 Kit (T cells) (Cat. No. 11462D)
Dynabeads FlowComp Mouse CD4+CD25+ Treg Cells Kit (Cat. No. 11463D)
Dynabeads FlowComp Mouse Pan T (CD90.2) Kit (Cat. No. 11465D)
Dynabeads Mouse Pan T (Thy1.2) (Cat. No. 11443D)
DETACHaBEAD Mouse CD4 Kit (T cells) (Cat. No. 12406D)
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The M stands for magnetic. M-280 refers to hydrophobic 2.8 micron beads, while M-270 refers to hydrophilic 2.8 micron beads. MyOne refers to 1 micron beads.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
Answering this question is not straightforward. It will depend on the detection method. When using HRP (horseradish peroxidase)-based detection system or radioactivity in combination with a good antibody, very little target is required. More target is required when using an AP (alkaline phosphatase)-based detection system. When a sensitive detection system is used, detection will most likely be in the nanogram range. In some cases, pictograms of target can be detected.
Find additional tips, troubleshooting help, and resources within our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
Within practical limits, the elution volume can be scaled up or down to suit your experiment. However, volumes less than 10 µL become more difficult to work with. In addition, the amount of target is important. If you have a lot of beads with a lot of bound target in a small elution volume, your elution may not be very efficient. Typically, 15-100 µL of beads may be eluted in 30 µL. For efficient recovery of the antigen and/or binding partners, the elution volume should at minimum equal the volume of the beads.
Find additional tips, troubleshooting help, and resources within our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
There are several methods to quantify the amount of antibody bound to the beads. The crudest method is to measure the concentration of antibody in the coupling reaction before and after antibody attachment. Either fluorescence measurements or absorbance at 280 nm can be used. Alternatively, you could measure the amount of antibody bound to the beads by fluorescence, chemiluminescence, or radiolabeling detection methods.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
Incubation time will depend on the immunogenicity of the primary antibody and its binding affinity with the specific antigens. For a good primary antibody, 30-40 minutes incubation should work well. If you are working with a poor antibody or a very low-abundance protein, you could try to increase binding by incubating overnight. However, this also increases the chance of background protein binding.
Find additional tips, troubleshooting help, and resources within our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
If the target protein has the same molecular weight as the heavy or light chain antibody, we would recommend covalently binding the antibody to the bead surface. This can be done by either crosslinking the antibody to the Dynabeads Protein A or G magnetic beads, or secondary coated beads, or by using one of the surface-activated Dynabeads magnetic beads.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
Using Dynabeads magnetic beads for protein isolation provides several advantages:
-Rapid binding kinetics: since the number of beads per volume for Dynabeads is approximately 1,000 times higher than for the same volume of a Sepharose slurry, the probability for Dynabeads magnetic beads to hit the target is far greater.
-Incubation time: due to the rapid binding kinetics, the protocol is usually very short.
-Low background: due to the rapid binding kinetics and the short incubation time, the background is also very low.
-Trapping of impurities: the beads offer no internal volume for binding or trapping of impurities.
-Low antibody consumption: this is because Dynabeads magnetic beads are nonporous, uniform superparamagnetic, monodispersed, highly crosslinked polystyrene microspheres consisting of an even dispersion of magnetic material throughout the bead. The beads are coated with a thin layer of a highly crosslinked polystyrene shell that encases the magnetic material and prevents any leakage from the beads or trapping of ligands in the bead interior. The outer layer also provides a defined surface area for the adsorption or coupling of various molecules such as antibodies. Uniformity of bead size and shape provide consistent physical and chemical properties. These uniform physical characteristics lead to high-quality, reproducible results.
-Reproducibility: due to easier practical handling, such as pipetting. No centrifugation steps or preclearing are required.
Find additional tips, troubleshooting help, and resources within ourProtein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
No. Not only is dithionite a reducing agent, but the strong affinity of the dithionite ion for bivalent and trivalent metal cations (M2+, M3+) allows it to enhance the solubility of iron, making it a chelating agent. As a result, the iron in the Dynabeads magnetic beads is reduced and pulled out when they are exposed to dithionite. The same is observed if Dynabeads magnetic beads are exposed to DTT and EDTA. With EDTA, we highly recommend checking the minimal amount of EDTA that your specific molecules would tolerate for binding to the Dynabeads, and if it will affect your specific application. For some applications, low concentrations of EDTA can be tolerated by Dynabeads. On the other hand, using 10 mM EDTA with heating affects the binding of biotin molecules to Dynabeads streptavidin.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
Yes, they are compatible with 6-8 M Urea when used during post-coupling steps.
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Dynabeads magnetic beads, being magnetic in nature are really not designed to be centrifuged. That being said, the beads themselves are compact, as the pores in the polymer matrix are filled with magnetic material and coated with a final outer polymer shell that will further add to the rigidity of the beads. Hence, pressure should theoretically not be a problem for the beads themselves, but the force exerted by the beads on surrounding cells in the pellet may be detrimental to the cells.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
Magnetic beads, unlike agarose beads, are solid and spherical, and antibody binding is limited to the surface of each bead. While magnetic beads do not have the advantage of a porous center to increase the binding capacity, they are significantly smaller than agarose beads (1 to 4 µm), which collectively gives them adequate surface area-to-volume ratios for optimum antibody binding.
High-power magnets are used to localize magnetic beads to the side of the incubation tube and out of the way to enable cell lysate aspiration without the risk of also aspirating immune complexes bound to the beads. Magnetic separation avoids centrifugation, which can break weak antibody-antigen binding and cause loss of target protein.
However, an issue with the use of magnetic beads is that bead size variations may prevent all beads from localizing to the magnet. Additionally, while immunoprecipitation using agarose beads only requires standard laboratory equipment, the use of magnetic beads for immunoprecipitation applications requires high-power magnetic equipment that can be cost-prohibitive. Read more about our Magnetic Immunoprecipitation Products (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/immunoprecipitation.html#products).
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
This depends on the bead size. In general, for M-450 Dynabeads, 1 mg/mL beads is approximately equivalent to 1.3 X 10E7 beads/mL; for M-280 Dynabeads, 1 mg/mL beads is approximately equivalent to 6.7 X 10E7 beads/mL.
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Whether cells will internalize the Dynabeads magnetic beads during culture will depend on the cell type. Due to the bead size (usually 4.5µm in diameter) Dynabeads magnetic beads will not be internalized into the endocytic pathway e.g., via clathrin coated pits. The clathrin coated pits are typically not more than 500 nm in size, which is far too small for endocytosis of the beads. However, if cells with phagocytic activities (e.g., monocytes/macrophages) are present, the Dynabeads magnetic beads will be phagocytosed into the phagolysosomes by these specialized cells. So it would really depend on the cell type.
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We offer several Dynabeads magnetic beads that can be used for either positive isolation (keep the target cells) or for depletion (remove the target cell from a sample) that does not include any release mechanism:
- Dynabeads magnetic beads for depletion: Using Dynabeads magnetic beads for depletion is a very fast, efficient and easy method. Use pre-coated Dynabeads magnetic beads or coat your own target antibody onto our secondary coated beads, add to any sample (e.g., whole blood, PBMC, buffy coat, tissue digests), incubate for 20 minutes with mixing, apply to a magnet for 2 minutes, and you have your cells depleted.
- Dynabeads magnetic beads for positive isolation for molecular downstream assays: Positive isolation of target cells without bead release can be used when the aim is downstream molecular studies such as DNA, RNA, or protein analysis. In these applications, the isolated cells can be lysed while the beads are attached to the cells, and the beads can be removed after cell lysis. If the bead presence is not a problem, you can also culture the cells with the beads on. In most cases the surface antigen will be internalized after 2-3 days, and then the beads will fall off since the beads are too big to be internalized by the endocytosis pathway.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
In general, the size of the Dynabeads magnetic beads is so large that they will not be internalized. The clathrin-coated pits are typically not more than 500 nm in size, which will be too small for Dynabeads magnetic beads to be internalized by endocytosis. However, if the target cells have phagocytic activities such as monocytes/macrophages, the Dynabeads magnetic beads could be internalized by phagocytosis.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
The CELLection kits contain Dynabeads magnetic beads coated with a DNA linker (either via streptavidin or via an antibody for cell isolation) and a DNase enzyme for bead release. After the beads capture the target cells (either directly or indirectly), the cells are released by DNAse treatment to cleave the linker. As a result, the target cells are released from the Dynabeads magnetic beads, but still linked with capture antibody.
We offer three Dynabeads CELLection cell isolation kits:
- CELLection Epithelial Enrich Kit (Cat. No. 16203) contains Dynabeads magnetic beads coupled with anti-EpCAM monoclonal antibody
- CELLection Biotin Binder Kit (Cat. No. 11533D) is used for positive isolation of target cells with your own biotinylated antibody
- CELLection Pan Mouse IgG Kit (Cat. No. 11531D) is used for isolation of target cells with your own mouse IgGs
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
The beads in the CELLection Epithelial Enrichment kits are coated with Ber-EP4, which binds with high affinity to the epithelial cell marker EpCAM (Epithelial Cell Adhesion Molecule). The Epithelial Enrich product does not positively isolate circulating tumour cells, but rather provides the scientist with a highly sensitive enrichment technique that can be applied directly to whole blood, bone marrow, or MNC samples. The product is based on the theory that in disease-free (or rather circulating tumor cell-free) individuals, epithelial cells are normally absent from the blood stream. The isolation of epithelial cells from blood samples is thus a good indication of circulating tumor cells, but this will have to be confirmed by downstream assays. In addition to epithelial cells, a number of leukocytes will also be isolated due to trapping or unspecific binding. These are, however, significantly smaller than the epithelial cells, and researchers in this field should easily be able to visually identify or distinguish epithelial (tumor) cell from these other cells co-isolated with the Epithelial Enrich beads. Some 10,000 or more leukocytes may be isolated along with your epithelial cells from a 5 mL blood sample. The isolated cells should then be subjected to a downstream investigation to (1) verify epithelial cells (e.g., immune-staining via cytokeratin or similar), followed by (2) further analysis of the enriched cells for tumour cell confirmation and characterization.
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The EpCAM antigen is an epithelial cell adhesion molecule that is abundantly expressed both on the majority of cells of epithelial origin, as well as epithelial-derived tumor cells. The Ber-EP4 (anti-EpCAM) antibody on the beads will thus recognize both normal and cancerous epithelial cells, with the assumption that no epithelial cells are normally present in circulating blood when isolating tumor cells from blood, mononuclear cell, or bone marrow samples.
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There are three methods to remove the Dynabeads magnetic beads from the isolated cells:
1. DETACHaBEAD Kits (positive isolation) - where the release agent is a polyclonal anti-Fab reagent outcompeting the binding of the bead-bound antibody on the cell, giving both antibody- and bead-free cells. Kits are available for Human CD4+ and CD8+ T cells, CD19+ B cells, and CD34+ hematopoietic stem cells.
2. CELLection Kits - where the release agent is a DNase enzyme, digesting the DNA-linker between the antibody and the bead, ultimately leading to bead-free cells. Kits are available for human EpCAM (Ber-EP4) epithelial cells and streptavidin (binding any biotinylated antibodies).
3. FlowComp Kits - where the release reagent is biotin that is out-competing the des-biotinylated antibody coupled onto the beads. Available for human and mouse whole T cells, the T cells subsets CD4+ and CD8+, and human monocytes.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
For positive cell isolation we recommend that you use 1-2 x 10E7 total PBMC per mL, and 1 x 10E7 Dynabeads magnetic beads per mL of sample. Another way of determining how many beads you need is to assume approximately 4 beads per target cell. If you want to deplete your sample of a certain target cell, we recommend using the same cell concentration as above (1-2 x 10E7 total PBMC per mL), but at least double the amount of beads to 2 x10E7 Dynabeads magnetic beads per mL of sample. Or alternatively, assume about 8-10 beads per target cell to be depleted.
Note: Most Dynabeads magnetic beads for cell separations are supplied at approximately 4 x 10E8 beads/mL, thus 1 x 10E7 beads is only 25 µL (please check the specific product manual or label for each product to confirm)
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The major advantages of using positive isolation are the high purity and yield. In addition, the initial sample prep is generally not required, since the cells can be isolated directly from any sample such as whole blood, bone marrow, or tissue digests. If using the primary coated beads without bead release, the procedure is very simple and fast; for example you can get isolation of cells directly from whole blood in as little as 25 minutes, which also leads to very high cell viability. A general disadvantages for any positive isolation method is a potential activation of the cells, depending on the function of the target epitope on the target cell. Note that the Dynabeads magnetic beads are not directly causing activation; it is the antibody-cell epitope interaction that causes this. When using positive isolation with a release mechanism, some loss of yield must be expected, since not all links between the cells and beads will be broken.
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Positive isolation is a method for separation of one specific target cell population from a sample. By using Dynabeads magnetic beads, magnetic particles coated with target-specific antibodies, the beads will bind only the cells with the matching surface antigen expressed on the target cells' surface. Two different Dynabeads magnetic beads - based positive isolation methodologies may be employed depending on the downstream assays. Since the beads themselves are quite large (from 1 µm to 4.5 µm in diameter) we don't recommended that you use bead-bound cells directly in any flow cytometer. Thus, if you have a cellular downstream assay and want to use a flow cytometer, we recommend using one of our positive isolation kits where a bead-release mechanism is available.
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No, the PBS must be Ca2+- and Mg2+-free as these divalent ions can lead to activation of complement and aggregation of cells. Aggregation of cells in the sample can severely reduce both yield and purity of the isolation. EDTA must also be added to the isolation buffer to minimize activation of complement and aggregation. Alternatively EDTA can be replaced by 0.6% sodium citrate.
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When cell purity is the most important criteria, target cell activation is not a concern, or when the downstream applications are isolation of RNA or gDNA, then positive isolation is recommended. When the most important criteria is to isolate cells with minimum disturbance, and where the downstream applications include cell culture, study cell function, morphology, and flow cytometry, then negative isolation is recommended (note that positive isolation of cells can also be used when the downstream applications include cell culture, flow cytometry, or studying cell function, but then a positive isolation with bead release from the cells are required, by using e.g., FlowComp products or the Positive Isolation Kits containing DETACHaBEAD products).
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Thaw cells in their cryovial in a 37 degrees C water bath until a small ice-clump is left. Transfer the cells gently to a fresh 10-15 mL tube immediately after the cells are thawed and add 10 mL 20% FCS/human serum in droplets to the cells while gentle pipetting. Avoid air bubbles. Work fast. Centrifuge the cells 200 X g, 8 minutes. Discard the supernatant. Resuspend in the appropriate buffer/media.
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In general, freezing medium (10% DMSO and 90% FCS) or Gibco Recovery Cell Culture Freezing Medium (Cat. No. 12648-010) work well. Some cells will always die during the freezing process. In addition, freezing and thawing will cause some cells to lyse. The protocol to freeze mammalian cells using Gibco Recovery medium is as follows:
1. Thaw Recovery Cell Culture Freezing Medium, mix well, and keep at 2-8 degrees C until use.
2. For suspension cells proceed to step 3. For adherent cells, gently detach cells from the substrate on which they are growing using a suitable dissociation reagent such as Gibco TrypLE reagent. Resuspend cells in the complete medium required for that cell type.
3. Transfer cell suspension to a sterile 15 mL centrifuge tube.
4. Determine the viable cell density and percent viability using a Countess Automated Cell Counter (similar automated or manual methods may be used) and calculate the required volume of Recovery Cell Culture Freezing Medium to give a final cell density of 1 X 10E6 to 1 X 10E7 cells/mL.
5. Centrifuge cell suspension at 100-200 x g for 5-10 minutes. Aseptically decant supernatant without disturbing the cell pellet. Note: Centrifugation speed and duration may vary depending on cell type.
6. Resuspend the cell pellet in (2- 8 degrees C) chilled Recovery Cell Culture Freezing Medium at recommended viable cell density for specific cell type (typically 1 X 10E6 cells/mL or greater).
7. Dispense aliquots of cell suspension (mix frequently to maintain a homogeneous cell suspension) into cryovials according to the manufacturer's specifications (i.e., 1.5 mL in a 2 mL cryovial).
8. Achieve cryopreservation in an automated or manual controlled rate freezing apparatus following standard procedures (approximately 1 degree C decrease per minute).
9. Transfer frozen cells to liquid nitrogen, (vapor phase); storage at -200 degrees C to -125 degrees C is recommended.
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Bone marrow needs to be washed and diluted prior to addition of Dynabeads magnetic beads to make the sample less viscous. Washing and DNase treatment is recommended for preparing bone marrow cells prior to cell isolation using Dynabeads magnetic beads:
- Mix 2 mL (10E7-10E8 cells) bone marrow with 2 ml PBS w/ 0.1% BSA + 0.6% Na-citrate.
- Centrifuge at 600 g for 8 min at 18-25 degrees C.
- Discard the supernatant and resuspend to 5 mL with PBS w/ 0.1% BSA + 1mM CaCl2 + 0.5 mM MgCl2.
- Add 600 Kunitz units DNase I (120 Kunitz units DNase I per milliliter).
- Incubate cells for 30 minutes at 18-25 degrees C with both gentle tilting and rotation.
- Centrifuge cell suspension for 8 minutes, 600 x g, at 18-25 degrees C.
- Discard supernatant and resuspend cell pellet in 5 mL PBS w/ 0.1% BSA.
- Centrifuge cell suspension for 8 minutes, 600 x g, at 18-25 degrees C.
- Discard supernatant and resuspend at 1 x 10E8 cells per milliliter in RPMI 1640 / 1% FCS
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Follow standard tissue preparations using enzymes and mechanical disruption to get a single-cell suspension. Eliminate large aggregates by sieving the digested cell suspension through a cell strainer or filter through a 30 µm filter. Disruption of tissue normally results in some cell death and release of DNA. Free DNA will impair cell capture, recovery, and purity. DNase I treatment is performed by incubating the cell suspension in PBS with 0.1% BSA + 1 mM CaCl2 + 0.5 mM MgCl2 and 120 Kunitz units DNase I per ml at 18-25 degrees C for 30 min. (For CELLection products, wash cells to remove DNase before adding the beads.)
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Mononuclear cells (MNC), also known as peripheral blood mononuclear cells (PBMC), are prepared from whole blood, buffy coat, bone marrow, or umbilical cord blood by density gradient separation. The following protocol can be used for standard MNC preparation for positive isolation or depletion protocols:
1. Collect blood sample with anticoagulant present (EDTA, ACD, heparin). Dilute peripheral blood 1 + 1, buffy coat 1 + 2, bone marrow 1 + 1 and umbilical cord blood 1 + 3 in PBS w/ 0.1% BSA + 0.6% Na-citrate or 2 mM EDTA.
2. Layer up to 35 mL of the diluted sample over 15 mL gradient medium (such as Ficoll or Lymphoprep solution) in a 50 mL tube.
3. Centrifuge for 400 x g for 30-40 minutes at 18-20 degrees C. If blood has been stored for more than 2 hours, increase centrifugation time by 10 min.
4. Collect MNC from the interface and transfer cells to a 50 mL tube.
5. Wash MNC three times with PBS w/0.1% BSA by centrifugation at 300 x g for 8 min at 2-8 degrees C.
6. Resuspend the cells to 1 x 10E7 cells per milliliter in PBS with 0.1% BSA and cool to 2-8 degrees C.
Note: MNC contain T cells (50%), B cells (5-10%), NK cells (5-10%), and monocytes (30%) without granulocytes and very few platelets.
For use with Untouched/negative isolation kits, the following protocol is recommended to obtain MNC prep with low platelet numbers and the highest possible purity:
Whole blood/buffy coat and bone marrow can be used as a starting material.
1. Dilute 10-18 mL blood/buffy coat with PBS w/ 0.1% BSA + 0.6% Na-citrate or 2 mM EDTA to a total volume of 35 ml at 18-25 degrees C.
2. Add the diluted blood/buffy coat on top of 15 mL of gradient medium (such as Lymphoprep or Ficoll solution).
3 .Centrifuge at 160 x g for 20 min at 20 degrees C. Allow to decelerate without braking.
4. Remove 20 mL of supernatant to eliminate platelets.
5. Centrifuge at 350 x g for 20 min at 20 degrees C. Allow to decelerate without braking.
6 .Recover MNC from the plasma/Lymphoprep solution interface and transfer the cells to a 50 mL tube.
7. Wash MNC once with PBS w/ 0.1% BSA by centrifugation at 400 x g for 8 min at 2-8 degrees C.
8. Wash MNC twice with PBS w/ 0.1% BSA by centrifugation at 225 x g for 8 min at 2-8 degrees C and resuspend the MNC at 1 x 10E8 MNC per milliliter in PBS w/ 0.1% BSA.
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Buffy coat, also known as leukocyte concentrate, is the middle fraction of an anti-coagulated blood sample that sits under the plasma and on the top of red blood cells after centrifugation of the sample without using a density gradient reagent such as Ficoll solution. Buffy coat contains both leukocytes and platelets and can be used as a source of this cellular material.
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Typically, one milliliter of adult human blood contains:
~5 x 10E9 red blood cells
~7 x 10E6 leukocytes
~3 x 10E8 platelets
In the 7 x 10E6 leukocyte fraction, there are:
4 x 10E5 monocytes
1 x 10E5 NK cells
Lymphocytes:
2 x 10E5 B cells
1 x 10E6 T cells (approx. 70% are CD4+ T cells and 30% are CD8+ T cells)
Granulocytes:
5 x 10E6 neutrophils
2 x 10E5 eosinophils
4 x 10E4 basophils
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This will depend on your application. As a guideline, the 4.5 micron beads are best used for cell isolation and activation/expansion. These larger beads have a higher magnetic mobility, they are roughly the same size as mammalian cells, and are less likely to be taken up by the cells. The smaller 1 micron beads and 2.8 micron beads are often used when isolating nucleic acids or proteins, or for immunoprecipitation. In negative cell isolation kits, one micron beads are often used because of their higher binding capacity per milliliter of beads and faster binding kinetics. With negative selection, cells taking up any beads will not be a problem as you want to look at the remaining cell population anyway. The 2.8 micron Dynabeads magnetic beads, coated with secondary antibodies, protein A or protein G, or streptavidin are also used for positive cell isolation with primary antibodies of your own choice, targeting specific cell-surface antigens.
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Three different sizes of Dynabeads magnetic beads are available: One micron beads (look for MyOne magnetic beads in the product name), 2.8 micron beads, and 4.5 micron beads. In general, the binding capacity per milliliter of beads and binding kinetics increases as the bead size reduces.
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Dynabeads magnetic beads are super-paramagnetic, meaning they only display magnetic characteristics when a magnet is present. As soon as the magnet is removed, the beads handle like a liquid and are easily dispersed in the sample tube. For cell isolation purposes, this has clear advantages as it allows for gentle handing and reduced stress to the cells. Secondly, the beads all have the same size and shape, with rapid liquid-phase reaction kinetics. The smooth surface of the beads results in less non-specific binding. These properties tend to reduce variability and allow you to get more reliable and reproducible results for your purifications and your analyses whether you are looking at cells or any other target molecule (RNA/DNA/proteins/protein complexes/organelles/exosomes etc.)
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Here are some suggestions:
- Make sure that pH of the RPMI + 1% FBS is not too high: DNase I works best between pH 7.0-7.4 (O2 exposure of RPMI will result in increased pH and the pH-indicator will turn blue).
- Do not use RPMI + 10% FBS during DNase I treatment. 10% FBS gives a lower cell yield than 1% FBS (might be caused by DNase I inhibitory factors in some batches of FBS).
- Make sure that the buffer contains Mg2+/Ca2+ required for DNase activity.
- DNase I must be treated gently. Vigorously stirring of the DNase solution can reduce the enzyme activity. Never vortex the DNAse solution.
- When selecting low numbers of cells, we recommend that you pre-coat tubes with RPMI + 10% FBS to reduce cell loss (cells are sticky and will easily attach to the tube wall during selection).
- DNase I has good activity at 20 degrees C, however, we recommend pre-warming the RPMI + 1% FBS to 37 degrees C before starting DNase I treatment because ‘room temperature' varies from lab to lab.
- After cells are incubated with DNase Releasing Buffer it is essential that the bead-cell complexes are thoroughly pipetted before the magnetic separation to provide mechanical disruption to the DNA linker. Failure to pipette the cells will affect cell yield.
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Here are some suggestions:
- Since the DSB-X biotin -streptavidin bond becomes stronger over time, do not increase the time of the release step longer than stated in the protocol. In some instances a shorter incubation time can lead to higher yield.
- After incubation with the releasing agent, we recommend that you resuspend the sample well by pipetting >10 times using a 1 mL pipette before adding the sample to the magnet.
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Here are some suggestions:
- Sample preparation is very important. All isolations must be performed on single-cell suspensions. For isolation of certain cell types (e.g., monocytes) directly from whole blood, we recommend that you wash the blood before isolation to remove interfering factors in the sample.
- An appropriate mixer must be used for all incubations where ‘mixing' is specified. Any mixer providing either tilting and rotation or end-over-end mixing can be used (e.g., HulaMixer Sample Mixer (Cat. No. 15920D)).
- If using FlowComp kits where the antibodies are added to the cells before adding the beads (indirect method), excess antibodies should be removed by washing prior to adding the beads.
- After incubation with the releasing agent, we recommend that you re-suspend the sample well by pipetting >10 times using a 1 mL pipette before adding the sample to the magnet.
- The recommended isolation buffer must be used for cell isolations. The PBS must be Ca2+ and Mg2+ free as these divalent ions can lead to activation of complement and aggregation of cells.
- Aggregation of cells in the sample can severely reduce both yield and purity of the isolation.
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