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View additional product information for Dynabeads™ Sheep-Anti Mouse IgG - FAQs (11031)
77 product FAQs found
请查看以下可能原因：
•溶液太粘稠。
•蛋白质间相互作用导致磁珠聚集。
尝试以下建议：
•延长分离时间（将管子留在磁力架上2-5分钟）。
•向裂解液中加入DNase I（约0.01 mg/mL）。
•将结合和/或清洗缓冲液中的Tween20浓度增加至约0.05%。
•向结合和/或清洗缓冲液中加入最多至20 mM 的β-巯基乙醇。
对于小于1 kb的生物素标记DNA，我们推荐使用Dynabeads M270链霉亲和素磁珠和MyOne C1磁珠。对于大于1kb的双链DNA分子，我们推荐Dynabeads KilobaseBINDER试剂盒。KilobaseBINDER试剂包括M-280链霉亲和素偶联的Dynabeads磁珠和一种含有专利的固定活化剂的结合液，可结合较长的生物素化DNA分子以进行分离。请点击以下链接(https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/napamisc/capture-of-biotinylated-targets/immobilisation-of-long-biotinylated-dna-fragments.html)，查看关于长的生物素化DNA片段分离的更多信息。
可以，Dynabeads磁珠可用于分离单链DNA。链霉亲和素Dynabeads磁珠能够以生物素化的DNA片段为靶标，通过使双链DNA变性，从而去除非生物素化链。链霉亲和素偶联的Dynabeads磁珠不会抑制任何酶活性。因此，可以在固相上直接对磁珠结合的DNA进行下一步处理。请点击以下链接(https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/napamisc/capture-of-biotinylated-targets/preparing-single-stranded-dna-templates.html)，查看关于单链DNA捕获的更多信息。
磁化率能够衡量磁珠向磁力架迁移的速度，其大小取决于铁含量和氧化铁的特性。Dynabeads磁珠的磁化率是指质量磁化率，单位可以是cgs单位/g或m^3/kg（国际单位制）。对于亚铁磁性和铁磁性物质，质量磁化率取决于磁场强度（H），这些物质的磁化强度与H不是线性关系，而是随着场强增加而趋于饱和。因此， Dynabeads磁珠的质量磁化率是在固定条件下由标准操作程序而测定的。我们产品目录中给出的质量磁化率是国际单位制。磁化率由从高斯（cgs、emu）单位向国际单位的转换，是通过“高斯系数（emu/g或cgs/g）x 4π x 10^-3”而实现的。所得单位也被称为合理化质量磁化率，与（国际单位制）无量纲磁化率单位有所区别。通常，质量磁化率可用来衡量在非均匀磁场中影响物体的力（Fz）。测定Dynabeads磁珠的质量磁化率时，首先对样本称重，然后将样本放置于已知强度的磁场中。随后，再次称重得到样本重量（F1），并与关闭磁场时样本的重量（F0）进行对比。使用下述公式计算磁化率：K x 10^–3 = [(F1-F0) x m x 0.335 x 10^6]，K表示质量为m的样本的质量磁化率。最后，将磁化率转换为国际单位制。
有多种不同的方法可以检测配体与磁珠结合，包括光密度（OD）检测、荧光标记和放射性标记。
对于OD检测，应在配体固定到磁珠上之前检测配体的OD值，并将其与包被后上清液中剩余的配体浓度进行比较。这样可以粗略检测有多少蛋白与磁珠结合。 实验方案： 1.将分光光度计设置到正确的波长。使用偶联缓冲液作为空白组。 2.检测偶联前溶液的吸光值。根据配体的加入量，可能需要进一步稀释以读取吸光值。 3.检测偶联后溶液的吸光值。也可能需要进一步稀释以读取吸光值。 4.计算偶联效率，以“蛋白质摄取量%”表示，如下所示：[(偶联前溶液的吸光值x D) – (偶联后溶液的吸光值x D)] x 100/(偶联前溶液的吸光值 x D)，D = 稀释倍数。 对于荧光标记，我们建议对配体结合量进行反向定量，即检测偶联上清液中剩余的配体量（与原始样本对比），而不是直接检测磁珠上的配体量。将标记的配体加入到磁珠中，并检测上清液中剩余多少配体（而不是结合到磁珠上的配体）。通过与开始时加入的总配体量相比，可以计算出结合到磁珠上的配体量。由于Dynabeads磁珠具有自发荧光，因此，我们不推荐直接检测与磁珠结合的配体的荧光，而是推荐这种间接方法。标记物可以是FITC/PE等。有些研究人员也成功使用了直接检测方法（采用流式细胞仪）。 在3种方法中，放射标记的灵敏度最高，但难度最大。该方法涉及到对配体的一部分进行放射性标记。在偶联前，使用示踪剂量的放射性标记的I-125，将其以一定比例与“冷”配体混合。使用闪烁（γ）计数器对磁珠进行检测，并将磁珠的cpm值与标准品对比，得到磁珠上配体的绝对量。 实验方案： 1.取出适量磁珠，并使用1 mL结合缓冲液清洗。 2.吸取适量人IgG，置于一个单独的管子中。 3.将人IgG与I-125标记的人IgG（30,000–100,000 cpm）混合。 4.使用结合缓冲液将人IgG与I-125标记的人IgG混合物稀释至100mL。 5.室温下孵育30分钟，使用闪烁计数器检测cpm值。 6.清洗磁珠（和包被层）4次，再次检测cpm值。 使用下述方程计算结合率%：（清洗后cpm值/清洗前cpm值）x100%。Dynabeads磁珠有3种尺寸：4.5 µm （M-450）、2.8 µm （M-270/M-280）和1 µm （MyOne beads）。其中最大尺寸的磁珠非常适合细胞等较大的目标，2.8 µm磁珠推荐用于蛋白质组学和分子研究，而最小的1 µm磁珠则适用于自动化处理。
一般来说，在加入配体包被磁珠时，短时间超声是减少磁珠聚集、确保磁珠获得最佳均一性的好方法。一旦目标分子结合到磁珠，就要加倍小心了，以防结合被破坏。链霉亲和素磁珠本身能够承受超声。超声5分钟是可以的，更长时间超声的影响还未被测试。关于链霉亲和素-生物素的相互作用可否被超声破坏目前也尚无相关信息。
只有未包被的环氧基或甲苯磺酰基活化的磁珠可根据需要使用70%乙醇进行清洗除菌。包被的磁珠不可灭菌。
Dynabeads磁珠是一种大小均一、无孔、超顺磁的、单分散的、高度交联的聚苯乙烯微球，整个磁珠由均匀分散的磁性材料构成。该磁性材料由磁赤铁矿(γ-Fe2O3)和磁铁矿(Fe3O4)的混合物组成。在Dynabeads磁珠M-280和M-450中，铁(Fe)分别占磁珠重量的12%和20%。Dynabeads磁珠表面覆盖有一层薄的聚苯乙烯外壳，将磁性材料包裹在内，可防止磁珠泄漏或在内部捕获配体。此外，该外壳也可避免目标分子直接接触铁，同时为每次实验提供特定的表面来吸附或偶联各种分子。
磁珠尺寸和形状均一，确保物理和化学性质稳定一致，进而提高实验结果的质量和可重复性。
Dynabeads磁珠分为3种不同尺寸：4.5 μm （M-450磁珠），2.8 μm （M-270/M-280磁珠）和1 μm （MyOne磁珠）。
可以。请参见此海报（https://tools.thermofisher.com/content/sfs/posters/Exosome-poster-ISEV-2013-Boston.pdf）。
此外，这里还有一些相关引用：
•Blood 91:2573 (1998)
•Science 289:444 (2000)
•J Physiol 537:537 (2001)
•Mol Cell Proteomics 12:587 (2013)
•Biol Reprod 81:717 (2009)
是的，基于不同的膜表面标志物所分离外泌小体中的蛋白表达谱之间会有明显差异。这一结论由Tauro等（http://www.ncbi.nlm.nih.gov/pubmed/23230278）的研究证实，该研究团队基于EpCAM或A33这两种膜表面标志物，从人癌细胞系的条件培养基中分离出两群明显不同的外泌小体。蛋白组学研究结果显示这两群外泌小体是独特的。
我们拥有多款外泌小体分离试剂盒，包括外泌小体—人源CD63（货号10606D），外泌小体—人源CD9（货号10614D），外泌小体—人源CD81（货号10616D）和外泌小体—人源EpCAM，适用于凭借这些常用的外泌小体膜表面抗原来实现外泌小体分离操作。如果您希望使用您的自备抗体通过其他特异性膜表面标志物来分离外泌小体，您也可使用我们的Dynabeads外泌小体免疫沉淀（ProteinA，货号10610D），Dynabeads外泌小体免疫沉淀（ProteinG，货号10612D）或用于分离/检测的外泌小体—链霉亲和素产品（货号10608D）。此外，用户也可选用抗小鼠IgG的Dynabeads磁珠（货号11031或11033）和识别特定膜表面标志物的小鼠单抗来实现外泌小体分离操作。
一般通过流式细胞仪（使用CD9、CD63、TSG101和Alix等膜表面标志物）来鉴定外泌小体，通过EM来研究其形态和尺寸，或凭借LC-MS/MS来实现更为详细的蛋白分析。
这些标志物要基于外泌小体的细胞来源进行选择。最常用于外泌小体分离和鉴定的膜表面标志物为CD9、CD63、CD81或TSG101。下表列举了一些最近用于鉴定或分离外泌小体的参考文献和膜表面标志物：
膜表面标志物
参考文献
Alix, CD63, EpiCam, HSP70, TSG101
Mol Cell Proteomics 12:587 (2013)
CD9, CD63
Hum Mol Genet 21:R125 (2012)
CD63, MHC II
J Biol Chem 278:52347 (2003)
CD9, CD81, Lamp1, TSG101
Cancer Res 67:7458 (2007)
CD63
Nature Cell Biol 9:654 (2007)
Alix, CD37, CD53, CD63, CD81, CD82, TSG101
J Cell Biol 200:373 (2013)
CD59, CD63, CD133, TSG101
FASEB J 23:1858 (2009)
除沉淀法之外，外泌小体还可通过超速离心或密度梯度分离法实现分离。用户也可使用靶向外泌小体标志物（例如人源的CD9、CD63、CD81、EpCAM）的Dynabeads磁珠或二抗包被的Dynabeads磁珠（使用靶向其他外泌小体膜表面标志物的不同抗体），凭借磁性方法来分离外泌小体。
外泌小体被报道具有多种不同的功能，如抗原呈递、凋亡、血管发生、炎症和凝血作用，这些作用是通过蛋白/脂类交换或信号途径的激活来实现的。外泌小体提供了一种胞间遗传物质交换的全新机制，并能够介导细胞间的相互通讯。外泌小体也能够转运和传播传染性物质，如朊蛋白和逆转录病毒。
外泌小体是微小的卵形或杯形膜结构，大小在30-150 nm，其中包含有mRNA，microRNA，蛋白和脂类。外泌小体可由正常，异常或肿瘤细胞释放进入血、尿、唾液和乳汁等体液中。外泌小体源于内吞型细胞器，并作为与质膜融合的多泡体（MVB）而从细胞释放出来(J Cell Biol 200:373 (2013)).
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。
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。）
Please review the following possibilities for why your Dynabeads magnetic beads are not pelleting:
- The solution is too viscous.
- The beads have formed aggregates because of protein-protein interaction.
Try these suggestions:
- Increase separation time (leave tub on magnet for 2-5 minutes)
- Add DNase I to the lysate (~0.01 mg/mL)
- Increase the Tween 20 concentration to ~0.05% of the binding and/or washing buffer.
- Add up to 20 mM beta-merecaptoethanol to the binding and/or wash buffers.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
For biotin-labeled DNA that is less than 1 kb, we recommend you use Dynabeads M270 Streptavidin (Cat. No. 65305) and MyOne C1 magnetic beads (Cat. No. 65001). We recommend our Dynabeads KilobaseBINDER Kit (Cat. No. 60101), which is designed to immobilize long (>1 kb) double-stranded DNA molecules. The KilobaseBINDER reagent consists of M-280 Streptavidin-coupled Dynabeads magnetic beads along with a patented immobilization activator in the binding solution to bind to long, biotinylated DNA molecules for isolation. Please see the following link (https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/napamisc/capture-of-biotinylated-targets/immobilisation-of-long-biotinylated-dna-fragments.html) for more information in regards to long biotinylated DNA fragment isolation.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
Yes, Dynabeads magnetic beads can be used to isolate single-stranded DNA. Streptavidin Dynabeads magnetic beads can be used to target biotinylated DNA fragments, followed by denaturation of the double-stranded DNA and removal of the non-biotinylated strand. The streptavidin-coupled Dynabeads magnetic beads will not inhibit any enzymatic activity. This enables further handling and manipulation of the bead-bound DNA directly on the solid phase. Please see the following link (https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/napamisc/capture-of-biotinylated-targets/preparing-single-stranded-dna-templates.html) for more information in regards to single-stranded DNA capture.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
Magnetic susceptibility is a measure of how quickly the beads will migrate to the magnet. This will depend on the iron content and the character of the iron oxide. The magnetic susceptibility given for the Dynabeads magnetic beads is the mass susceptibility, given either as cgs units/g or m^3/kg (the latter being an SI unit). For ferri- and ferromagnetic substances, the magnetic mass susceptibility is dependent upon the magnetic field strength (H), as the magnetization of such substances is not a linear function of H but approaches a saturation value with increasing field. For that reason, the magnetic mass susceptibility of the Dynabeads magnetic beads is determined by a standardized procedure under fixed conditions. The magnetic mass susceptibility given in our catalog is thus the SI unit. Conversion from Gaussian (cgs, emu) units into SI units for magnetic mass susceptibility is achieved by multiplying the Gaussian factor (emu/g or cgs/g) by 4 pi x 10^-3. The resulting unit is also called the rationalized magnetic mass susceptibility, which should be distinguished from the (SI) dimensionless magnetic susceptibility unit. In general, magnetic mass susceptibility is a measure of the force (Fz) influencing an object positioned in a nonhomogenous magnetic field. The magnetic mass susceptibility of the Dynabeads magnetic beads is measured by weighing a sample, and then subjecting the sample to a magnetic field of known strength. The weight (F1) is then measured, and compared to the weight of the sample when the magnetic field is turned off (F0). The susceptibility is then calculated as K x 10^-3 = [(F1-F0) x m x 0.335 x 10^6], where K is the mass susceptibility of the sample of mass m. The susceptibility is then converted to SI units.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
There are different methods to check binding of ligands to the beads, including optical density (OD) measurement, fluorescent labeling, and radioactive labeling.
For OD measurement, you would measure the OD of the ligand before immobilization to the beads and compare it with the ligand concentration that is left in the supernatant after coating. This gives a crude measurement of how much protein has bound to the beads.
Protocol:
1.Set spectrophotometer to the right wavelength. As a blank, use the Coupling Buffer.
2.Measure the absorbance of the Pre-Coupling Solution. A further dilution may be necessary to read the absorbance, depending upon the amount of ligand added.
3.Measure the absorbance of the Post-Coupling Solution. A dilution may be necessary to read the absorbance.
4.Calculate the coupling efficiency, expressed as the % protein uptake, as follows. [(Pre-Coupling Solution x D) - (Post-Coupling Solution x D)] x 100/(Pre-Coupling Solution x D) where D = dilution factor.
For fluorescent labeling, we suggest negatively quantifying the amount of ligand bound by measuring ligand remaining in the coupling supernatant (compared to the original sample), rather than directly measuring the ligands on the beads. Add labeled ligand to the beads, and measure how much ligand is left in the supernatant (not bound to the beads). By comparing this with the total amount added in the first place, you can then calculate how much of the ligand that has been bound to the beads. Keep in mind that the Dynabeads magnetic beads are also autofluorescent, which is why direct measuring of fluorescence of the bead-bound ligands is not recommended, but rather this indirect approach. The label could be, for example, FITC/PE. Some researchers perform a direct approach with success (using a flow cytometer).
Radioactive labeling is the most sensitive method of the three, but it is also the most difficult one. It involves radioactively labeling a portion of the ligand. We use radiolabeled I-125 in tracer amounts and mix it with "cold" ligands in a known ratio before coupling. The absolute quantities for the ligand on the beads should be obtained by measuring the beads in a scintillation (gamma) counter and comparing the cpm with a standard.
Protocol:
1.Take out an appropriate amount of beads and wash the beads in 1 mL of binding buffer.
2.Pipette out desired amount of human IgG in a separate tube.
3.Mix the human IgG with I-125-labeled human IgG (30,000 - 100,000 cpm).
4.Dilute the mixture of human IgG and I-125-labeled human IgG to 100 mL in binding buffer.
5.Incubate for 30 minutes at room temperature and measure the cpm in a scintillation counter.
6.Wash the beads (with coating) four times, and measure cpm again.
The % binding is calculated by using the equation : (cpm after washing/cpm before washing)x100%.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
Dynabeads magnetic beads come in three sizes: 4.5 µm (M-450), 2.8 µm (M-270/M-280), and 1 µm (MyOne beads). The largest of the Dynabeads magnetic beads is ideal for big targets like cells. The 2.8 µm beads are recommended for proteomics and molecular applications. The smallest of the beads, 1 µm, are ideal for automated handling.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
In general, short sonication is a good way to reduce aggregation of the beads and ensure optimal homogenous conditions at the time of ligand addition when coating the beads. When target is bound to the beads, more care is needed, as the binding might break. The streptavidin beads themselves should tolerate sonication. We have not tested sonication for long periods, but 5 minutes is fine. We do not have information about the streptavidin-biotin interaction being broken by such treatment.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
If desired, the uncoated epoxy or tosylactivated beads can be sterilized by washing with 70% ethanol. Coated beads cannot be sterilized.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
Dynabeads magnetic beads are uniform, non-porous, superparamagnetic, monodispersed and highly cross-linked polystyrene microspheres consisting of an even dispersion of magnetic material throughout the bead. The magnetic material within the Dynabeads magnetic beads consists of a mixture of maghemite (gamma-Fe2O3) and magnetite (Fe3O4). The iron content (Fe) of the beads is 12% by weight in Dynabeads magnetic beads M-280 and 20% by weight in Dynabeads magnetic beads M-450. The Dynabeads magnetic beads are coated with a thin polystyrene shell which encases the magnetic material, and prevents any leakage from the beads or trapping of ligands in the bead interior. The shell also protects the target from exposure to iron while providing a defined surface area for the adsorption or coupling of various molecules.
Uniformity of bead size and shape provides consistent physical and chemical properties. These uniform physical characteristics lead to high-quality, reproducible results.
The Dynabeads magnetic beads are available in three different sizes: 4.5 µm (M-450 beads), 2.8 µm (M-270/M-280 beads) and 1 µm (MyOne 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.
The secondary-coated Dynabeads magnetic beads can be coupled to a primary antibody by a direct or an indirect approach.
Direct approach: The Dynabeads magnetic beads are first coupled with your primary antibody and then used for isolating your target cell type.
Indirect approach: The cells are first incubated with your primary antibody(ies). The Dynabeads magnetic beads are then added to the antibody-coated target cells.
Secondary-coated Dynabeads magnetic beads can be used in several cell isolation approaches:
Cell depletion--using an antibody to target the unwanted cell type and a secondary-coated Dynabeads magnetic beads product.
Negative cell isolation--using a cocktail of antibodies to target all unwanted cell types and a secondary-coated Dynabeads magnetic beads product (using the indirect approach).
Positive cell isolation without detachment--using an antibody to target the wanted cell type and a secondary-coated Dynabeads magnetic beads product.
Positive cell isolation with detachment--using an antibody to target the wanted cell type and a CELLection Dynabeads magnetic beads product.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
You may use one of our secondary-coated, surface-activated, or streptavidin-coated Dynabeads magnetic beads, and coat it with a primary antibody to target your cell type.
The Dynabeads magnetic beads product you choose will depend on the primary antibody available for cell targeting and the downstream application for the isolated cells:
-For primary antibodies made in mouse, use the CELLection magnetic beads Pan Mouse IgG Kit, Dynabeads magnetic beads Goat Anti-Mouse IgG, Dynabeads magnetic beads Pan Mouse IgG, Dynabeads magnetic beads Rat Anti-Mouse IgM, Dynabeads magnetic beads Rat Anti-Mouse IgM, or Dynabeads magnetic beads Sheep-Anti Mouse IgG
-For primary antibodies made in rat, use the Dynabeads magnetic beads Sheep Anti-Rat IgG
-For primary antibodies made in rabbit, use the Dynabeads magnetic beads M-280 Sheep Anti-Rabbit IgG
-For primary antibodies made in any species, use the CELLection magnetic beads Biotin Binder Kit, Dynabeads magnetic beads Biotin Binder, Dynabeads magnetic beads FlowComp Flexi, Dynabeads magnetic beads M-450 Epoxy, or Dynabeads magnetic beads M-450 Tosylactivated
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Yes, a cocktail of primary antibodies can be added to a cell suspension in order to pull out several target cell populations with one secondary-coated Dynabeads magnetic beads product.
The Dynabeads magnetic beads Pan Mouse IgG (110.41; 110.42) works very well with a cocktail of mouse IgGs for the simultaneous capture of multiple cell types. it is recommended that you use an indirect technique with antibody cocktails (add all Ab to cells, wash off excess Ab, then add beads to capture Ab-coated cells).
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
The indirect technique is chosen when the antigen targeted by the primary antibody is expressed in low density on the target cell surface. This is due to the fact that free antibodies will find their target antigen more easily than antibodies linked to the Dynabeads magnetic beads. Also when using the indirect technique, an excess of free antibody can be added to the system, allowing ample opportunity for monoclonals to find the target antigen. Finally, an indirect technique can be useful when a cocktail of monoclonal antibodies is used to deplete unwanted cells during negative isolation of a cell type. This is because antibodies against all unwanted cell types can be added at once to the starting cell population, provided the antibodies are from one species. The antibody-coated cells can then be targeted with secondary-coated Dynabeads magnetic beads.
The direct technique is chosen when a limiting amount of monoclonal antibody is needed for targeting the cells of interest during positive isolation or depletion (e.g., when the target antigen is present at high density). It can also help when the possibility of interaction from the secondary antibody needs to be avoided, or if a stock preparation of primary coated Dynabeads magnetic beads is desired. Additionally the direct technique can be used when you do not want to cover all antigen sites with antibody (e.g., when you want to analyze the isolated cells by flow cytometry).
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
When a secondary-coated Dynabeads magnetic beads product is used for negative isolation or depletion of cells you can choose between the following techniques.
Direct technique
-Add primary IgG antibody to the secondary-coated Dynabeads magnetic beads with specificity to the species of your primary IgG antibody
-Add the resulting primary coated Dynabeads magnetic beads to cells for capture and separation
Benefits:
-You can make up a stock of beads
-Use less primary antibody
-Save time, fewer steps and is easier to optimize than the indirect procedure
-Lower background, additional steps increase the chance of nonspecific signal
-Lower cost, since direct procedures require much lower amounts of tag specific antibodies than indirect procedures
OR
Indirect technique
-Add primary IgG antibody to the cells
-Wash cells to remove excess, unbound antibody
-Add secondary-coated Dynabeads magnetic beads with specificity to the species of your primary IgG antibody to capture primary antibody coated cells
Benefits:
-You can use a cocktail of antibodies to coat different cell types i.e., a negative isolation approach
-Can be used when target antigen expression is low for more efficient binding of antibody to cells.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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.
The Dynabeads Co-immunoprecipitation Kit is specially designed for protein complex pulldown only, not for simple IP. These beads are the Dynabeads M270 Epoxy (Cat. No. 14301) beads and are used for covalent binding of the antibody so it will not be co-eluted off with the target complex during mild elution. The kit also contains a buffer system that should make it easier to optimize for recovering the complex.
With the Dynabeads Protein A or G magnetic beads, there is only a non-covalent binding between the Protein G on the beads and the antibody. Thus, the antibody will be co-eluted with the target protein unless you crosslink it to the beads beforehand. In general, we recommend the Dynabeads Co-immunoprecipitation Kit for complex pulldown, especially if you are working with large, labile complexes or if your downstream application is mass spectrometry.
Secondary coated Dynabeads magnetic beads (like the Dynabeads anti-mouse IgG beads) can also be used for IP, but, the drawback when compared to Dynabeads Protein A or G magnetic beads, is that they will only work with an antibody from 1 species. However, the binding between the primary antibody and the secondary antibody might be a bit stronger than between Protein A or G and the antibody. Hence, you can apply more stringent washing conditions. That being said, Dynabeads Protein A and G magnetic beads should give a very low background.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis 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.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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.
Yes. See this poster (https://tools.thermofisher.com/content/sfs/posters/Exosome-poster-ISEV-2013-Boston.pdf).
In addition, here are some citations:
- Blood 91:2573 (1998)
- Science 289:444 (2000)
- J Physiol 537:537 (2001)
- Mol Cell Proteomics 12:587 (2013)
- Biol Reprod 81:717 (2009)
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Yes, exosomes isolated with different surface markers can be distinctive in their protein profile. This has been demonstrated by Tauro et al. (http://www.ncbi.nlm.nih.gov/pubmed/23230278), who isolated two distinctive populations of exosomes based on surface markers EpCam or A33 from conditioned cell culture medium from a human carcinoma cell line. This proteomics study indicated that these two populations of exosomes are unique.
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We have exosome isolation kits for Exosome-Human CD63 (Cat. No. 10606D), Exosome-Human CD9 (Cat. No. 10614D), Exosome-Human CD81 (Cat. No. 10616D), and Exosome-Human EpCAM intended for isolating exosomes with these commonly used exosome surface antigens. If you are interested to isolating exosomes with other specific surface markers using your own antibody, you can use our Dynabeads exosome immunoprecipitation (Protein A, Cat. No. 10610D), Dynabeads exosome immunoprecipitation (Protein G, Cat. No. 10612D), or Exosome-Streptavidin for isolation/detection (Cat. No. 10608D). In addition, anti-mouse IgG Dynabeads magnetic beads (Cat Nos. 11031 or 11033) also can be employed in exosome isolation using mouse monoclonal antibodies against selected surface markers.
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Exosomes are usually characterized by flow cytometry (using surface markers such as CD9, CD63, TSG101, and Alix), by EM to study morphology and size, or by detailed protein analysis by LC-MS/MS.
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It depends on the cell source from which the exosomes were derived. The most commonly used surface markers for isolating and characterizing exosomes are CD9, CD63, CD81, or TSG101. Here are some of the recent references and surface markers for identifying or isolating exosomes:
Alix, CD63, EpiCam, HSP70, TSG101 Mol Cell Proteomics 12:587 (2013)
CD9, CD63 Hum Mol Genet 21:R125 (2012)
CD63, MHC IIJ Biol Chem 278:52347 (2003)
CD9, CD81, Lamp1, TSG101 Cancer Res 67:7458 (2007)
CD63 Nature Cell Biol 9:654 (2007)
Alix, CD37, CD53, CD63, CD81, CD82, TSG101J Cell Biol 200:373 (2013)
CD59, CD63, CD133, TSG101 FASEB J 23:1858 (2009)
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Exosomes can be isolated by ultracentrifugation or density gradient separation in addition to a precipitation approach. Exosomes can also be isolated by a magnetic approach using Dynabeads magnetic beads targeting exosome markers such as Human CD9, CD63, CD81, EpCAM or secondary antibody-coated Dynabeads magnetic beads using different antibodies against other exosomal surface markers.
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A range of different functions have been reported such as antigen presentation, apoptosis, angiogenesis, inflammation, and coagulation by protein/lipid exchange or activation of a signaling pathway. Exosomes provide a novel vehicle for genetic exchange between cells and mediate cell to cell communication. Exosomes also transport and propagate of infectious cargoes such as prion and retrovirus.
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Exosomes are small, membrane-bound ovoid to cup shaped particles around 30-150 nm in size containing mRNA, microRNA, proteins, and lipids. Exosomes are released by normal, abnormal, or neoplastic cells into body fluid such as blood, urine, saliva, and breast milk. Exosomes originate from the endocytic compartment and are released from cells as multivesicular bodies (MVB) fused with plasma membrane (J Cell Biol 200:373 (2013)).
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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.
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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.
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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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