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View additional product information for CELLection™ Pan Mouse IgG Kit - FAQs (11531D)
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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
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：
•在CELLection Dynabeads 磁珠的包被过程中，对靶标抗体的数量进行滴定/优化。
•如果目的细胞的数量很低，或细胞表面的靶标抗原浓度很低，则推荐使用间接法技术。
•使用>1 x 10E7个磁珠/mL样品（>25 μL）。
•在使用前对全血样本进行洗涤。
•为了确保获得最高效率的细胞释放效果，绝对不要在溶解冻干DNA酶的过程中涡旋DNA酶溶液。
•在释放细胞的过程中，请将新鲜配制的RPMI/1% FCS预热至37°C。
•确保缓冲液的pH值在7.0-7.4之间以获得理想的DNA酶I活性。更高的pH值会抑制DNA酶的活性。
•RPMI中应含有足量的Mg2+以帮助DNA酶发挥活性。
•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：
•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。
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.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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
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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.
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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:
- Titrate/optimize the target antibody amount when coating the CELLection Dynabeads magnetic beads.
- If the target cell number is low or the target antigen concentration on the cell surface is low, use the indirect technique.
- Always use >1 x 10E7 beads per milliliter sample (>25 µL).
- Wash whole blood before use.
- To ensure the most efficient cell release, never vortex DNase when resuspending freeze-dried DNAse.
- For cell release, use fresh RPMI/1% FCS pre-warmed to 37 degrees C.
- Check the pH of the RPMI. It should be 7.0-7.4. Higher pH will inhibit DNase activity.
- RPMI should contain sufficient Mg2+ for DNase activity.
- 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:
- 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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