Dynabeads™ Human T-Activator CD3/CD28 免疫磁珠使用方法
Protocols
This product allows for easy physiological activation of human T cells, without the need for preparing antigen-presenting cells (APCs) or antigen.
Dynabeads Human T-Activator CD3/CD28提供一种人T细胞的简便激活方法,无需APCs或抗原。
Preparations
See lifetechnologies for recommended Dynabeads products for positive or negative isolation of all human T cells, or specific T cell subsets.
Prepare cell culture medium
准备
人T细胞、T细胞亚群正选或负选的推荐产品参见*。
准备细胞培养基。
Dynabeads Washing Procedure
Dynabeads should be washed before use.
1 Resuspend the Dynabeads Human T-Activator CD3/CD28 in the vial.
2 Transfer the desired volume of Dynabeads to a tube.
3 Add an equal volume of Buffer, or at least 1 ml, and mix (vortex for 5 seconds, or keep on a roller for at least 5 min).
4 Place the tube on a magnet for 1 min and discard the supernatant.
5 Remove the tube from the magnet and resuspend the washed Dynabeads in the same volume of Culture Medium as the initial volume of Dynabeads taken from the vial (step 2).
Dynabeads洗涤步骤
Dynabeads使用前需要清洗。
1.小瓶重悬Dynabeads。
2.转移需要体积到试管。
3.添加等体积缓冲液,或至少1mL,混匀(漩振5s或roller 5min)。
4.试管在磁极放置1min,弃掉上清。
5.从磁极拿出,用等体积培养基重悬Dynabeads。
Activation of Human T Cells
1 Start with 8 × 104 purified T cells in 100-200 μl medium in a 96-well tissue culture plate.
2 Add 2 μl Dynabeads Human T-Activator CD3/CD28 to obtain a bead-to-cell ratio of 1:1.
3 Incubate in a humidified CO2 incubator at 37°C, according to your specific experimental requirements.
4 Harvest the activated T cells and use directly for further analysis.
5 For flow cytometry applications, remove the beads prior to staining. Place the tube on a magnet for 1-2 minutes to separate the beads from the solution. Transfer the supernatant containing the cells to a new tube.
活化人T细胞
1. 8 × 104 purified T cells+100-200 μl 培养基,放于96孔培养板。
2. 添加2 μl Dynabeads Human T-Activator CD3/CD28。细胞磁珠比为1:1。
3. 根据实验需求,CO2 培养箱37°C 孵育。
4. 收获活化T细胞,用于下游分析。
5. 进行流式细胞分析时,染色前移除磁珠。试管在磁极放置1-2min,以从溶液分离磁珠。转移包含细胞的上清液到新试管。
Expansion of Human T Cells
1 Start with 1-1.5 × 106 purified T cells/ml in culture medium in a suitable tissue culture plate or tissue culture flask.
2 Add Dynabeads Human T-Activator CD3/CD28 at a bead-to-cell ratio of 1:1.
3 Add 30 U/ml rIL-2.
4 Incubate in a humidified CO2 incubator at 37°C, according to your specific experimental requirements.
5 Examine cultures daily, noting cell size and shape. Cell shrinking and reduced proliferation rate is typically observed in exhausted cell cultures.
6 Count the cells at least twice weekly after thorough re-suspension.
7 When the cell density exceeds 2.5 × 106 cells/ml or when the medium turns yellow, split cultures back to a density of 0.5-1 × 106 cells/ml in culture medium containing 30 U/ml rIL-2.
人T细胞扩增
1.T 细胞培养液密度:1-1.5 × 106。
2.添加Dynabeads Human T-Activator CD3/CD28 磁珠。磁珠细胞比为1:1。
3.添加30 U/ml rIL-2。
4.根据实验需求,CO2 培养箱37°C 孵育。
5.每日观测,注意细胞大小和形状变化。耗竭的细胞培养物细胞会缩小,扩增也减少。
6.重悬后每周至少做2次细胞计数。
7.细胞密度超过2.5 × 106 cells/ml或培养基变黄,稀释回1-1.5 × 106cells/ml,含30 U/ml rIL-2。
Re-Stimulation
Cell cultures showing signs of exhaustion (typically at day 7-10 of expansion) can be re-stimulated several times by adding fresh Dynabeads Human T-Activator CD3/CD28 and rIL-2. The CD8+ T cells remain cytotoxic after repeated re-stimulations. Re-stimulation is typically necessary when cell shrinking and a reduced rate of proliferation is observed. Do not use an excess volume of Dynabeads Human T-Activator CD3/CD28, as excess Dynabeads per cell may inhibit expansion. Prior to re-stimulation, remove the used Dynabeads by transferring the cells to a suitable tube. Place the tube on a magnet for 1-2 minutes until the Dynabeads have moved to the side of the tube. Transfer the supernatant containing the cells to a new tube. Continue as described below.
1 Count the cells and resuspend to a density of 1 × 106 cells/ml in culture medium with 30 U/ml rIL-2 in a suitabl culture plate or tissue culture flask.
2 Add Dynabeads Human T-Activator CD3/CD28 at a bead-to-cell ratio of 1:1.
3 Add 30 U/ml rIL-2.
4 Incubate in a humidified CO2 incubator at 37°C for the length of your specific experiment.
5 Examine cultures daily, noting cell size and shape. Cell shrinking and reduced proliferation rate is typically observed in exhausted cell cultures.
6 Count the cells at least twice weekly after thorough re-suspension.
7 When the cell density exceeds 2.5 × 106 cells/ml or when the medium turns yellow, split cultures back to a density of 0.5-1 × 106 cells/ml in culture medium with 30 U/ml rIL-2.
重刺激
扩增后7-10天,细胞培养会发生耗竭,通过重刺激可以恢复。
1.细胞计数,重悬到1 × 106 cells/ml,含30 U/ml rIL-2。
2.添加Dynabeads Human T-Activator CD3/CD28 磁珠。磁珠细胞比为1:1。
3.添加30 U/ml rIL-2。
4.根据实验需求,CO2 培养箱37°C 孵育。
5.每日观测,注意细胞大小和形状变化。耗竭的细胞培养物细胞会缩小,扩增也减少。
6.重悬后每周至少做2次细胞计数。
7.细胞密度超过2.5 × 106 cells/ml或培养基变黄,稀释回1-1.5 × 106cells/ml,含30 U/ml rIL-2。
使用方法英文版源Dynabeads产品。中文版由红荣微再编辑翻译。
Dynabeads™ Human T-Activator CD3/CD28 免疫磁珠
产品英文名 Dynabeads™ Human T-Activator CD3/CD28 for T Cell Expansion and Activation
产品中文名 Dynabeads™ Human T-Activator CD3/CD28 免疫磁珠
产品介绍 Dynabeads® Human T-Activator CD3/CD28 用于活化和扩增人 T 细胞和 T 细胞克隆,以作研究之用。Dynabeads® CD3/CD28 T Cell Expander 提供了活化和扩增 T 细胞的简单方法,无需抗原呈递细胞或抗原。 Dynabeads® Human T-Activator CD3/CD28 是均匀的 4.5 µm 超顺磁珠,与抗 CD3 和抗 CD28 单克隆抗体的优化混合物偶联。 通过使抗 CD3 和抗 CD28 抗体与 Dynabeads® 结合,这种微珠可以提供 T 细胞活化和扩增所需的原始和共刺激信号。
Dynabeads免疫磁珠
品牌 | 货号 | 产品描述 | 包装 | 价格 |
Thermo | 11131D | Dynabeads™ Human T-Activator CD3/CD28 磁珠 | 2 mL | 8385元 |
Thermo | 11132D | Dynabeads™ Human T-Activator CD3/CD28 磁珠 | 5 x 2 mL | 26006元 |
Thermo | 11161D | Dynabeads™ Human T-Activator CD3/CD28 磁珠 | 0.4 mL | 1575 元 |
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