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提供一種人T細胞的簡便激活方法，無需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

準備

人T細胞、T細胞亞群正選或負選的推薦產(chǎn)品參見*。
準備細胞培養(yǎng)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洗滌步驟

Dynabeads使用前需要清洗。

1.小瓶重懸Dynabeads。

2.轉(zhuǎn)移需要體積到試管。

3.添加等體積緩沖液，或至少1mL，混勻（漩振5s或roller 5min）。

4.試管在磁極放置1min，棄掉上清。

5.從磁極拿出，用等體積培養(yǎng)基重懸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細胞

1.  8 × 10⁴ purified T cells+100-200 μl 培養(yǎng)基，放于96孔培養(yǎng)板。

2. 添加2 μl Dynabeads Human T-Activator CD3/CD28。細胞磁珠比為1:1。

3. 根據(jù)實驗需求，CO₂ 培養(yǎng)箱37°C 孵育。

4. 收獲活化T細胞，用于下游分析。

5. 進行流式細胞分析時，染色前移除磁珠。試管在磁極放置1-2min，以從溶液分離磁珠。轉(zhuǎn)移包含細胞的上清液到新試管。

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細胞擴增

1.T 細胞培養(yǎng)液密度：1-1.5 × 10^6。

2.添加Dynabeads Human T-Activator CD3/CD28 磁珠。磁珠細胞比為1:1。

3.添加30 U/ml rIL-2。

4.根據(jù)實驗需求，CO₂ 培養(yǎng)箱37°C 孵育。

5.每日觀測，注意細胞大小和形狀變化。耗竭的細胞培養(yǎng)物細胞會縮小，擴增也減少。

6.重懸后每周至少做2次細胞計數(shù)。

7.細胞密度超過2.5 × 10⁶ cells/ml或培養(yǎng)基變黃，稀釋回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

擴增后7-10天，細胞培養(yǎng)會發(fā)生耗竭，通過重刺激可以恢復。

1.細胞計數(shù)，重懸到1 × 10⁶ cells/ml，含30 U/ml rIL-2。

2.添加Dynabeads Human T-Activator CD3/CD28 磁珠。磁珠細胞比為1:1。

3.添加30 U/ml rIL-2。

4.根據(jù)實驗需求，CO₂ 培養(yǎng)箱37°C 孵育。

5.每日觀測，注意細胞大小和形狀變化。耗竭的細胞培養(yǎng)物細胞會縮小，擴增也減少。

6.重懸后每周至少做2次細胞計數(shù)。

7.細胞密度超過2.5 × 10⁶ cells/ml或培養(yǎng)基變黃，稀釋回1-1.5 × 10⁶cells/ml，含30 U/ml rIL-2。

使用方法英文版源Dynabeads產(chǎn)品。中文版由紅榮微再編輯翻譯。

Dynabeads™ Human T-Activator CD3/CD28 免疫磁珠

產(chǎn)品英文名 Dynabeads™ Human T-Activator CD3/CD28 for T Cell Expansion and Activation

產(chǎn)品中文名 Dynabeads™ Human T-Activator CD3/CD28 免疫磁珠

產(chǎn)品介紹  Dynabeads® Human T-Activator CD3/CD28 用于活化和擴增人 T 細胞和 T 細胞克隆，以作研究之用。Dynabeads® CD3/CD28 T Cell Expander 提供了活化和擴增 T 細胞的簡單方法，無需抗原呈遞細胞或抗原。 Dynabeads® Human T-Activator CD3/CD28 是均勻的 4.5 µm 超順磁珠，與抗 CD3 和抗 CD28 單克隆抗體的優(yōu)化混合物偶聯(lián)。 通過使抗 CD3 和抗 CD28 抗體與 Dynabeads® 結(jié)合，這種微珠可以提供 T 細胞活化和擴增所需的原始和共刺激信號。

Dynabeads免疫磁珠

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