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View additional product information for Dynabeads™ Human T-Expander CD3/CD28 - FAQs (11141D)
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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）。
天然情况下，细胞表面的TCR-CD3复合物会持续内吞和重新插入胞膜。当Dynabeads 磁珠与培养细胞上的CD3/CD28相结合时，TCR-CD3复合物即会发生内吞并随之降解。当这一进程发生时，胞膜表面不再有磁珠结合的靶点，因此磁珠就被释放到培养基中。磁珠—细胞聚集物能够存在3-4天，直至磁珠从细胞上脱落。
T细胞受体（TCR-CD3）复合物由数个不同的亚基所组成，其中TCR的可变免疫球蛋白区（V）与配体间发生结合。CD3亚基和TCR的胞内尾部会与胞浆信号蛋白发生相互作用。复合物的胞内部分存在有几种内吞信号。
组成型内吞（无配体结合条件）：
我们从文献中得知，约有20种基于酪氨酸的内吞模体和2种双亮氨酸模体能够在无配体结合的条件下持续介导TCR-CD3复合物的胞膜内吞过程。再循环过程约为100分钟（从胞膜运送至早期内涵体，再返回胞膜），10次循环之后TCR-CD3复合物会靶向进入溶酶体中的降解途径。
诱导性内吞（存在配体结合条件）：
TCR-CD3复合物的配体依赖型内吞过程是由泛素介导的。一旦结合了配体，泛素即与复合物胞内尾部的某一氨基酸发生偶联，从而导致复合物的网格蛋白依赖型内吞及随后的溶酶体降解过程（内吞在2小时左右到达平台期 (Nature 375:148 (1995))
当磁珠与CD3-CD28结合时，复合物将发生主动的内吞和降解过程。当这一进程发生时，胞膜表面不再有磁珠结合的靶点，因此磁珠就被释放到培养基中。如果不将磁珠从培养基中移除，它们则会在TCR-CD3复合物重新出现在胞膜表面时再次与其结合。
参考培养T细胞的经验，起始种板密度为~0.5–1 x 10E6个细胞/mL，并在浓度达到~1.5–2.5 x 10E6个细胞/mL时进行传代。
我们推荐您在添加了2 mM 谷氨酰胺和10%FBS，以及2000 U/mL小鼠rIL-2（也可使用人源的rIL-2）的advancedRPMI-1640中培养小鼠T细胞。磁珠:靶细胞比例为2:1。对于扩增小鼠Treg细胞的应用，我们推荐您在适当的培养板/培养瓶中以1-1.5 x 10E6/mL T细胞开始尝试。
我们推荐您在添加了2 mM 谷氨酰胺和10%FBS，以及30 U/mL小鼠rIL-2（也可使用人源的rIL-2）的advancedRPMI-1640中培养小鼠T细胞。最佳的磁珠:靶细胞比例为1:1。对于短时程活化小鼠T细胞的操作，我们推荐在96孔培养板中每孔100-200 μL培养基的条件下，以每孔8 x 10E4个小鼠T细胞开始尝试。对于扩增小鼠T细胞的应用，我们推荐您在适当的培养板/培养瓶中以1-1.5 x 10E6/mL T细胞开始尝试。磁珠:细胞比率及IL-2浓度与激活T细胞时相同。
Dynabeads Human T-Activator CD3/CD28专为初始T细胞的活化进行了优化，而Dynabeads Human Treg Expander专为调节性T细胞进行了优化。这两款产品拥有相同的抗体克隆和相同的CD3:CD28比例。
是的，所有的T细胞激活后都会发生CTLA-4的表达上调现象。不过，在使用Dynabeads 磁珠激活T细胞的过程中，不存在CTLA-4（B7）的配体，所以T细胞的扩增不会受到抑制。与之相反，表达B7的APC会与CTLA-4相结合，从而产生抑制性信号。
Dynabeads CD3/CD28/CD137专为抗原特异性/primedT细胞的活化进行了优化，能够近距离提供较弱的抗CD3信号和抗CD28与抗CD137抗体的协同刺激信号。在活化和扩增记忆型T细胞时，我们推荐使用1:10的磁珠:靶细胞比例。在primed/抗原特异性T细胞的扩增中，每个T细胞使用过多的磁珠可能会诱导凋亡。与之相反，Dynabeads CD3/CD28（无CD137）产品专为初始T细胞的活化进行了优化；与抗原特异性/primedT细胞相比，初始T细胞的活化需要更强的抗CD3活化信号。
通常情况下我们推荐在第8-12天之间使用新磁珠进行重复刺激，基于您的应用而定。请按照产品手册中的说明进行重复刺激操作。
不，不会结合。该产品是特异性的抗CD28抗体。
可以，当T细胞与Dynabeads 磁珠孵育时，它们通常会在12小时内在孔中形成肉眼可见的细胞—磁珠聚集物。2-3天后细胞将进入大量扩增阶段（细胞分裂），这时它们的尺寸会发生显著增加，形态也会变得更加伸展。扩增之后（第8-12天），它们会恢复其通常的形态（圆形和清晰的边界）。
在培养3-4天后，大多数Dynabeads 磁珠会从细胞上脱落下来。
在扩增实验方案中，我们推荐您在第8-12天的时候（时间视具体实验而定），借助于磁力架来去除磁珠。彻底吹打磁珠:细胞复合物，之后放置于磁力架上2分钟，再将含有释放的细胞的上清液移至新的离心管中。弃去用过的磁珠，加入新鲜的磁珠进行二次刺激。
在短时程活化中，可直接对磁珠结合的细胞进行裂解，以用于后续分子水平的检测。第1-3天时，磁珠会与细胞形成强烈的聚集，因此在这一时期很难在不损失大量细胞的条件下去除磁珠。
不会，只有T细胞会响应刺激信号并在培养条件下生长。一周后CD3+细胞的纯度通常会超过95%。刺激MNC会产生某些问题，因为系统中存在的单核细胞可能会吞噬磁珠。我们建议您去除MNC中的单核细胞，或在T细胞活化/扩增之前通过冻存/复苏，塑料粘附或使用Dynabeads CD14（货号11149D，适于人源RUO）等标准方法对T细胞进行分离。
这要具体视您使用的产品而定：
•CTS Dynabeads CD3/CD28（货号40203D）和Dynabeads Human T-Expander CD3/CD28（货号11141D）：在T细胞的扩增操作中（>3天）使用3:1的磁珠:T细胞比例。重复刺激时请使用1:1的磁珠:细胞比例。
•Dynabeads Human and Mouse T Cell Activation CD3/CD28（货号11131D、11132D、11161D、11456D、11452D、11453D）：短时程刺激，扩增和重复刺激时，请使用1:1的磁珠:细胞比例。
•Dynabeads Human Treg Expander（货号11129D）：进行细胞扩增时请使用4:1的磁珠:细胞比例。
•Dynabeads Human T-Activator CD3/CD28/CD137（货号11162D，11163D）：用于T细胞克隆/细胞系的扩增和重复刺激时，请使用1:5-10的磁珠:细胞比例(1:10)。
Dynabeads CD3/CD28专为初始T细胞的激活进行了优化，能够在近距离给予强烈的抗CD3信号和抗CD28抗体的协同刺激信号。刺激完成后，T细胞中的大部分会成为表达CD62L、CD45RO、CD28、CD27、CCR7和CD95的T stem central memory/T central memory cells （中央记忆型T细胞）。
其中只有一小部分为效应记忆型T细胞或终末分化（TEMRA CD57+）细胞，而不同于由抗CD3抗体（OKT-3）扩增的T细胞。
使用Dynabeads CD3/CD28磁珠刺激的CD4+ T辅助细胞将会分化为不同的T辅助细胞亚群，具体视体外培养条件下添加的细胞因子而定。
•Dynabeads + IL-2的组合能够诱导形成Th1亚群（也可添加IL-12）
•Dynabeads + IL-4 + IL-2的组合能够诱导形成Th2亚群
•Dynabeads + IL-1b + IL-6，IL-21和TGFβ将诱导形成Th17亚群
T细胞的激活过程在数分钟内即会发生。何时对细胞进行分析基于您的实验而定（如T细胞信号：数分钟内；体外扩增：8–12天）。
一些CD8+ T细胞并不表达CD28抗原。这意味着在操作CD8+ T细胞克隆时——与操作CD4+细胞克隆相反——必须确定CD8+细胞克隆是否表达CD28。
- 对于CD8+细胞的多克隆性扩增操作，请按照产品手册中的描述，在激活和重复刺激过程中使用推荐的磁珠：细胞比率。不要在第8-12天之前进行重复刺激，刺激时间视具体应用而定（形成很多细胞:磁珠聚集物，细胞产生大量对生长十分重要的IL-2，因此在这一时间点不应更换培养基）。另一个推荐的选项是在重复刺激过程中向培养基中添加20-100U IL-2，但这一操作不是必需的。
- 对于抗原特异性/primed人源CD8+细胞而言，我们推荐您使用Dynabeads Human T-Activator CD3/CD28/CD137产品，CD137有助于这些细胞生长和增殖地更好；或使用仅包被了CD3和CD28抗体的Dynabeads 磁珠。请参见以下参考文献（http://onlinelibrary.wiley.com/doi/10.1111/j.1365-3083.2011.02564.x/pdf）。
我们推荐您使用人AB血清（从AB血型的供体提取的血清，不含抗A或抗B抗体）。也可使用常规的FBS，但T细胞克隆扩增研究中的大多数研究者已换成AB血清。
所有T细胞激活或扩增试剂盒中磁珠的尺寸均为4.5μm。
Dynabeads CD3/CD28能够提供比传统方法（如丝裂原或可溶性/平板结合抗体）更高的扩增效率和更稳定的结果。这主要是因为：
•优化的磁珠—抗体比率和稳定的磁珠包被效果
•磁珠的大小与哺乳动物细胞很接近，因此能够为靶T细胞上的TCR/CD3和CD28提供三维的和同步的信号从而真实地模拟APC
•Dynabeads CD3/CD28提供简单易用的即用型形式，从生产日期开始有24个月的保质期
•一个能涵盖小鼠和人类的研究，翻译研究，和临床应用的扩增平台
•激活初始T细胞可诱导出更多中央记忆型表型 (Cytotherapy 16:619 (2014)).
在天然条件下，T细胞的活化是由树突细胞等抗原呈递细胞（APC）上的主要组织相容性复合物肽和B7家族成员对T细胞上的T细胞受体（TCR）/CD3和CD28的募集作用来介导的。这两种刺激信号对于产生有效的免疫反应均是必需的。第一信号途径能够确保只有含特异性识别该肽的TCR的T细胞被激活。初始T细胞持续表达的唯一协同刺激受体为CD28，该第二信号途径使T细胞能够对一个抗原进行响应。在缺少CD28协同刺激的条件下，只有T细胞受体信号则导致T细胞无能。
包被了CD3/CD28抗体的Dynabeads 磁珠可作为人工APC（aAPC），来模拟T细胞的天然激活过程。
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。）
这里为用户提供一些保持细胞最优生长条件的提示：
•保持0.5– 1.5 x 10E6个细胞/mL的细胞最佳生长密度非常重要。当细胞密度超过1.5-2.5 x 10E6个细胞/mL时，就需要重新进行刺激。
•请按照产品手册中提供的磁珠:细胞比例，每8-12天重新刺激一次。
•请按照手册中所提供每一产品特定的正确磁珠:细胞比例进行活化操作。使用过量磁珠会抑制细胞活化/扩增。
•使用优化的细胞培养基，加入：
◦生长因子（IL-2，IL-7，IL-15，或其他细胞因子）
◦促生长添加剂（如人血清/FBS，L-谷氨酰胺/Glutamax，自由基清除剂（10 mM N-乙酰半胱氨酸））
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.
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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Superparamagnetic means that the Dynabeads magnetic beads exhibit magnetic properties when placed within a magnetic field, but have no residual magnetism when removed from the magnetic field.
This means that your targeted cells, proteins, or nucleic acids are only subjected to magnetic forces during the time the beads are on the magnet. The beads do not aggregate, but remain evenly dispersed in suspension.
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Yes. The antibodies are covalently bound and should be very stable in your applications.
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Depending on the antibody coated on the Dynabeads magnetic beads, the shelf life can vary from 24-36 months.
Some kits may have 18 months shelf life depending on other components supplied in the kit. The kits are guaranteed for 6 months from when you receive them.
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These two kits use different antibody clones which have different affinities for CD3 or CD28 antigen. The ratio of CD3/CD28 antibodies and bead concentrations are different as well in the two kits. Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation (Cat. No. 11131D) is mainly designed for T cell activation and Dynabeads Human T-Expander CD3/CD28 (Cat. No. 11141D) is mainly designed for T cell expansion.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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).
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In nature there is a constitutive internalization and re-entry of the TCR-CD3 complex to the cell surface. When Dynabeads magnetic beads bind to CD3/CD28 on the cells in culture, the TCR-CD3 complex is internalized and then degraded. When this happens the beads do not have any target on the surface of the cells to bind to and therefore the beads will be released into the medium. The bead-cell clusters last for about 3-4 days until the beads fall off the cells.
The T-cell receptor (TCR-CD3) complex consists of several different subunits where the variable immunoglobulin domains of TCR (V) bind to the ligand. The cytoplasmic tails of the CD3 subunits and TCR interact with cytosolic signaling proteins. In this cytosolic part of the complex several types of internalization signals are located.
Constitutive internalization (without ligand binding): From the literature we understand that approximately 20 tyrosine-based internalization motifs and 2 di-leucine motifs will constitutively remove the TCR- CD3 complex from the plasma membrane without ligand binding. The recycling time is approximately 100 minutes (from plasma membrane to early endosomes and back to plasma membrane), and after 10 such cycles the TCR- CD3 complex is targeted for degradation in lysosomes.
Induced internalization (with ligand binding): The ligand-dependent internalization of the TCR-CD3 complex is a ubiquitin-dependent internalization process. Upon ligand binding, ubiquitin is coupled to a certain amino acid in the cytosolic tail of the complex, which results in a clathrin-dependent internalization of the complex followed by lysosomal degradation (the internalization reaches plateau around 2 hours (Nature 375:148 (1995)). When the beads bind to CD3-CD28, the complex will actively be internalized and degraded. When this happens the beads do not have any target on the surface of the cells to bind to and therefore the beads will be released into the medium. If the beads are not removed from the medium they can re-bind to the TCR- CD3 complex when they re-appear at the plasma membrane.
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The rule of thumb is to seed the T cells at a concentration of ~0.5-1 x 10E6 cells/mL and to split the T cells when the concentration is ~1.5-2.5 x 10E6 cells/mL.
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We recommend that you culture mouse T cells in advanced RPMI-1640 with 2 mM glutamine and 10% FBS supplemented with 2000 U/mL mouse rIL-2 (human rIL-2 could also be used). The bead-to-cell ratio is 2:1. We recommend that you start with 1-1.5 X 10E6/mL T cells in suitable culture plate or flask for expanding mouse Treg cells.
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We recommend that you culture mouse T cells in advanced RPMI-1640 with 2 mM glutamine and 10% FBS supplemented with 30 U/mL mouse rIL-2 (human rIL-2 can also be used). The optimal bead-to-target cell ratio is 1:1. For short-term activation of mouse T cells, we recommend starting with 8 x 10E4 mouse T cells/well in 100-200µL medium in 96-well culture plates. For expansion of mouse T cells, we recommend that you start with 1-1.5 x 10E6/mL T cells in suitable culture plate or flask. The bead-to-cell ratio and concentration of IL-2 is the same as for activating T cells.
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The protocol for Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation (Cat. No. 11131D) is optimized for the activation of naive T cells, whereas the protocol for Dynabeads Human Treg Expander (Cat. No. 11129D) is optimized for regulatory T cell expansion. Both products have the same beads and contain the same antibody clones in the same CD3:CD28 ratio.
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Yes, all T cells up-regulate CTLA-4 upon activation. However, when using Dynabeads magnetic beads for activation of T cells, no ligand for CTLA-4 (B7) is present, so T cell expansion is not inhibited. In contrast, APCs express B7 that binds CTLA-4 and cause an inhibitory signal.
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Dynabeads Human T-Activator CD3/CD28/CD137 (Cat. Nos. 11162D, 11163D) is optimized for activation of Ag-specific/primed T cells by giving a weak anti-CD3 signal and co-stimulatory signals with the anti-CD28 and anti-CD137 antibodies in close proximity. For activation and expansion of memory T cells, we recommend 1 bead per 10 target cells. For expansion of primed/antigen-specific T cells, too many beads per T cell may induce apoptosis. In contrast, the Dynabeads CD3/CD28 (without the CD137) products are optimized for activation of naive T cells which require a stronger anti-CD3 activation signal than Ag-specific/primed T cells.
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Generally we recommend re-stimulating with fresh beads between days 8-12 depending on your application. Follow the product manual for instructions on re-stimulation.
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No, it does not. It is a specific anti-CD28 antibody.
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Yes, when T cells are incubated with Dynabeads magnetic beads, they typically form visible cell-bead clusters in the wells within 12 hours. After 2-3 days the cells enter the blast stage (cell division) where they increase considerably in size and their morphology changes to a more stretched appearance. After expansion (days 8-12), they will regain their usual morphology of round and clearly defined cells.
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After 3-4 days in culture, most of the Dynabeads magnetic beads have detached from the cells.
For expansion protocols, we recommend that you remove the beads with the help of a magnet between days 8-12 depending on the assay. Pipette the bead:cell complexes thoroughly before applying to the magnet for 2 minutes, and transfer the supernatant with the released cells to a new tube. Discard the used beads and add fresh beads for re-stimulation.
For short-term activation, the cells can be lysed while they are still on the beads for molecular assays. Between days 1-3, the beads form strong clusters with the cells, thus it is hard to remove the beads in this period without losing a lot of the cells.
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No, only T cells will get the stimulation signal and grow in culture. After a week the purity of CD3+ cells is typically above 95%. Stimulating MNC can be problematic due to the presence of monocytes as they will phagocytose the beads. We recommend that you remove the monocytes from the MNC or to isolate T cells before T cell activation/expansion by standard methods such as freeze/thaw, plastic adherence, or use of Dynabeads CD14 (Cat. No. 11149D, for human RUO).
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This depends on the product you are using:
- Dynabeads Human T-Expander CD3/CD28 (Cat. No. 11141D): For T cell expansion (>3 days) use 3 beads per T cell (3:1). For re-stimulation use 1 bead per cell (1:1).
- Dynabeads Human and Mouse T Cell Activation CD3/CD28 (Cat. Nos. 11131D, 11132D, 11161D, 11456D, 11452D, 11453D): For short-termstimulation, expansion, and re-stimulation, use 1 bead per T cell (1:1).
- Dynabeads Human Treg Expander (Cat. No. 11129D): For expansion use 4 beads per T cell (4:1).
- Dynabeads Human T-Activator CD3/CD28/CD137 (Cat. No. 11162D, 11163D): For expansion and re-stimulation of T cell clones/cell lines use 1 bead per 5 - 10 T cells (1:10).
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Dynabeads CD3/CD28 is optimized for activation of naive T cells by giving a strong anti-CD3 signal and a co-stimulation signal with anti-CD28 antibody in close proximity. After stimulation, the majority of the T cells are T stem central memory/T central memory cells expressing CD62L, CD45RO, CD28, CD27, CCR7 and CD95.
Only a small fraction of the cells are effector memory T cells or terminal differentiated (TEMRA CD57+) cells, in contrast to T cells expanded with anti-CD3 antibody (OKT-3).
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CD4+ T helper cells stimulated with Dynabeads CD3/CD28 will differentiate into different T helper subsets, depending on the cytokines added during in vitro culture conditions.
- Dynabeads + IL-2 give a Th1 subset (IL-12 may also be added)
- Dynabeads + IL-4 + IL-2 give a Th2 subset
- Dynabeads +IL-1b + IL-6, IL-21 and TGFβ give a Th17 subset
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The activation of T cells happens within minutes. When you analyze the cells depends on your assay (e.g., T cell signaling: within minutes, for in vitro expansion: 8-12 days).
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Some CD8+ T cells don't express the CD28 antigen. This means that when working on CD8+ T cell clones, as opposed to CD4+ clones, it is essential to determine if the CD8+ clones are expressing CD28.
- For polyclonal expansion of CD8+, use the recommended number of beads per cell for activation and re-stimulation as described in the product manual. Do not re-stimulate before day 8-12 depending on the application (very strong cell:bead clusters are formed, and the cells are producing a lot of IL-2, which is important for growth, so the media should not be changed at this point). Another optional recommendation is to use 20-100 U IL-2 in the medium when re-stimulating, but this not critical.
- For antigen-specific/primed human CD8+ cells, we recommend using the Dynabeads Human T-Activator CD3/CD28/CD137; CD137 helps these cells to grow and expand better and Dynabeads magnetic beads coated with CD3 and CD28 antibodies only. Please see the following reference (http://onlinelibrary.wiley.com/doi/10.1111/j.1365-3083.2011.02564.x/pdf).
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We recommend using AB human serum (serum taken from donors with AB blood type that do not have antibodies against A or B). Regular FBS can also be used, but most people switch to AB serum for T cell clone expansion studies.
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Bead size for all of the T cell activation and expansion kits is 4.5 µm.
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Dynabeads CD3/CD28 offers higher expansion rate and more consistent results than traditional methods (e.g., mitogens or soluble/plate-bound antibodies). This is due to:
- Reproducible coating of optimized antibody ratios on the beads
- The bead size is close to mammalian cells, thus truly mimicking the APC to provide three-dimensional and simultaneous signals to TCR/CD3 and CD28 on the target T cells.
- Dynabeads CD3/CD28 offer a ready-to-use format that is simple and easy to use and have 24 months shelf life from the manufacturing date.
- An expansion platform covering mouse and human research, translational research, and clinical applications
- Activates naive T cells into a more central memory phenotype (Cytotherapy 16:619 (2014)).
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In nature, activation of T cells occurs through the engagement of both the T cell receptor (TCR)/CD3 and CD28 on the T cell by the major histocompatibility complex peptide and B7 family members on the antigen-presenting cells (APC) such as dendritic cells. Both of these two stimulating signals are required for production of an effective immune response. The first signal ensures that only a T cell with a TCR specific to that peptide is activated. The only co-stimulatory receptor expressed constitutively by naive T cells is CD28, this second signal licenses the T cell to respond to an antigen. In the absence of CD28 co-stimulation, T cell receptor signaling alone results in anergy.
Dynabeads magnetic beads coated with CD3/CD28 antibodies are artificial APCs (aAPC) and mimic the natural activation of T cells.
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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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Here are a few tips to keep the optimal cell culture growth conditions:
- It is important to keep the cell density at 0.5-1.5 x 10E6 cells/mL for optimal growth. When the cell density exceeds 1.5-2.5 x 10E6 cells/mL, re-stimulation is required.
- Re-stimulate every 8-12 days with bead:cell ratio according to the product manual.
- Activate with the correct bead:cell ratio for each specific product according to the manual. Too many beads can inhibit cell activation/proliferation.
- Use optimized cell culture media, add:
-- Growth factors (IL-2, IL-7, IL-15, or other cytokines)
-- Growth enhancing supplements (e.g., human serum/FBS, L-glutamine/glutamax, radical scavenger (10 mM N-acetyl cysteine
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