人CD3/CD28 T细胞激活磁珠可以简单快捷的活化及扩增 T 细胞，无需饲养层细胞（抗原递呈细胞）和抗原。磁珠为直径5 µm的惰性超顺微球，同时共价偶联抗 CD3 和抗 CD28 抗体，模拟抗原呈递细胞的作用。细胞培养过程中可使用重组人IL-2 刺激 T 细胞扩增。细胞活化或扩增后，磁珠可以通过磁力架去除。
适用范围
人外周血单核细胞（PBMC）或 T 细胞亚群：包括CD3⁺ T 细胞、CD4⁺ T 细胞或CD8⁺ T 细胞。
实验试剂
1. 清洗 buffer
含有 2 mM EDTA 和 2% 胎牛血清（FBS）的 PBS，或者含有 2 mM EDTA 和 0.5% BSA 的 PBS，需预先通过 0.22 μm 滤膜过滤除菌。
2. 细胞培养基
RPMI1640 + 10% FBS + 2 mM 谷氨酰胺（可选加入 100 U/mL 青链霉素双抗）。当需要扩增 T 细胞时，向培养基内加入 30 U/mL 重组人 IL-2。此外，商品化 T 细胞培养基同样适用，培养基内添加的生长因子可根据实验要求进行调整。
注意事项
取用 T 细胞激活磁珠前彻底重悬磁珠（如涡旋 >30 秒，或置于旋转仪 5 分钟），吹打时避免产生气泡。
进行流式检测前，使用磁力架去除磁珠（可以通过轻柔吹打释放结合在细胞上的磁珠），收集上清液，进行下一步的流式检测。
操作流程示例（以分选后人 CD3⁺ T 细胞为例）：
1. 清洗磁珠
a. 彻底重悬试管中的磁珠（如涡旋 >30 秒，或置于旋转仪 5 分钟）。
b. 吸取目标体积的磁珠至流式管中，加入同等体积的清洗 Buffer。若不足 1 mL，添加 1 mL 的清洗 Buffer，涡旋 5 秒，或使用移液器吹打混匀，注意避免产生气泡（此处也可直接使用细胞培养基清洗）。
c. 将流式管置于磁力架上 3 分钟，弃上清。
d. 重复步骤 b‑c，本次使用细胞培养基对磁珠再次清洗。共清洗磁珠两次。
e. 使用细胞培养基重悬磁珠（例如：吸取 25 μL 磁珠进行清洗，清洗后用 1 mL 细胞培养基进行重悬）。
2. 人 T 细胞的激活（以 96 孔板为例）
a. 调整 T 细胞浓度为 1×10⁷ cells/mL，每孔中加入 25 μL T 细胞，此时孔内含有 2.5×10⁵ 个 T 细胞，保持培养基体积为 100 μL。
b. 向孔中加入 100 μL 清洗后重悬的磁珠，此时磁珠和细胞的比例为 1:1（可根据实验需要自行调整磁珠和细胞的使用比例，推荐比例为磁珠:细胞 = 1:1）。
c. 将细胞与磁珠置于 37℃，5% CO₂ 培养箱中孵育，根据实验需要决定细胞的培养时长。
d. 收获激活的 T 细胞，用于下游实验分析。
3. 人 T 细胞的扩增
a. 调整 CD3⁺ T 细胞种板浓度为 1‑1.5×10⁶ cells/mL，按照磁珠与细胞比例 1:1 加入激活磁珠。
b. 激活 2 天后，向培养基中添加 30 U/mL 的重组人 IL‑2。
c. 细胞与磁珠放于 37℃，5% CO₂ 培养箱中孵育，根据实验需要决定细胞的培养时长。
d. 每日查看细胞激活与扩增情况，注意细胞的大小和形状。当细胞皱缩或增殖速度明显放慢时，提示细胞可能耗竭。
e. 推荐在细胞激活的前 2 天不要处理细胞，2 天后随时观察细胞状态，当培养基变黄或孔内细胞数目过多时换液或传代。每次换液或传代后，应及时补充重组人 IL‑2 至 30 U/mL。
f. 定期进行细胞计数，当细胞密度超过 2.5×10⁶ cells/mL 时，轻柔吹打混匀，将细胞密度调整为 0.5‑1×10⁶ cells/mL。
激活效果
使用人 CD3/CD28 T 细胞激活磁珠，刺激 CD3⁺ T 细胞 24 到 48 小时，细胞用 FITC anti‑human CD69 抗体（克隆号 FN50）和 PE anti‑human CD25 抗体（克隆号 BC96）标记，进行流式细胞仪分析。刺激前后 CD25⁺ CD69⁺ T 细胞占总细胞的比例分别为 0.70% 和 75.81%。
Human CD3/CD28 T Cell Activation Magnetic Beads provide a simple and rapid method for activating and expanding T cells without the need for feeder cells (antigen‑presenting cells) or antigens. The beads are inert, superparamagnetic microspheres of 5 µm diameter, covalently coupled with anti‑CD3 and anti‑CD28 antibodies to mimic the function of antigen‑presenting cells. During cell culture, recombinant human IL‑2 can be used to stimulate T cell expansion. After activation or expansion, the beads can be removed using a magnetic separator.
Applications
Human peripheral blood mononuclear cells (PBMCs) or T cell subsets, including CD3⁺ T cells, CD4⁺ T cells, or CD8⁺ T cells.
Experimental Reagents
Washing Buffer
PBS containing 2 mM EDTA and 2% fetal bovine serum (FBS), or PBS containing 2 mM EDTA and 0.5% BSA. The buffer must be pre‑filtered through a 0.22 μm filter.
Cell Culture Medium
RPMI1640 + 10% FBS + 2 mM glutamine (100 U/mL penicillin‑streptomycin may be added optionally). When T cell expansion is required, add 30 U/mL recombinant human IL‑2 to the medium. Commercial T cell culture media are also suitable, and growth factors added to the medium can be adjusted according to experimental requirements.
Precautions
Thoroughly resuspend the T cell activation beads before use (e.g., vortex for >30 seconds or place on a rotator for 5 minutes). Avoid generating bubbles during pipetting.
Before flow cytometry analysis, remove the beads using a magnetic separator (gently pipetting can release beads bound to cells), collect the supernatant, and proceed with flow cytometry.
Protocol Example (Using Purified Human CD3⁺ T Cells as an Example)
1. Washing the Beads
a. Thoroughly resuspend the beads in the tube (e.g., vortex for >30 seconds or place on a rotator for 5 minutes).
b. Transfer the required volume of beads to a FACS tube and add an equal volume of Washing Buffer. If the bead volume is less than 1 mL, add 1 mL of Washing Buffer, vortex for 5 seconds, or mix by pipetting (avoid bubbles). Cell culture medium can also be used directly for washing.
c. Place the FACS tube on a magnetic separator for 3 minutes and discard the supernatant.
d. Repeat steps b‑c, this time washing the beads with cell culture medium. Wash the beads twice in total.
e. Resuspend the beads in cell culture medium (for example: take 25 μL of beads for washing, then resuspend in 1 mL of cell culture medium after washing).
2. Activation of Human T Cells (Using a 96‑Well Plate as an Example)
a. Adjust the T cell concentration to 1×10⁷ cells/mL. Add 25 μL of T cells per well, giving 2.5×10⁵ T cells per well, and keep the culture volume at 100 μL.
b. Add 100 μL of washed and resuspended beads to each well, resulting in a bead‑to‑cell ratio of 1:1 (the ratio can be adjusted according to experimental needs; the recommended ratio is beads:cells = 1:1).
c. Incubate the cells and beads in a 37°C, 5% CO₂ incubator for the desired length of time.
d. Harvest activated T cells for downstream analysis.
3. Expansion of Human T Cells
a. Adjust the CD3⁺ T cell plating concentration to 1‑1.5×10⁶ cells/mL and add activation beads at a bead‑to‑cell ratio of 1:1.
b. Two days after activation, add recombinant human IL‑2 to the culture medium at 30 U/mL.
c. Incubate the cells and beads in a 37°C, 5% CO₂ incubator for the desired length of time.
d. Check cell activation and expansion daily, paying attention to cell size and shape. When cells shrink or proliferation slows significantly, it may indicate exhaustion.
e. It is recommended not to disturb the cells during the first 2 days of activation. After 2 days, monitor cell status regularly. Change medium or subculture when the medium turns yellow or the cell number per well becomes too high. After each medium change or subculture, replenish recombinant human IL‑2 to 30 U/mL.
f. Count cells regularly. When the cell density exceeds 2.5×10⁶ cells/mL, gently pipette to mix and adjust the cell density to 0.5‑1×10⁶ cells/mL.
Activation Performance
Human CD3⁺ T cells were stimulated with Human CD3/CD28 T Cell Activation Beads for 24 to 48 hours. Cells were stained with FITC anti‑human CD69 antibody (clone FN50) and PE anti‑human CD25 antibody (clone BC96) and analyzed by flow cytometry. The proportion of CD25⁺ CD69⁺ T cells among total cells before and after stimulation was 0.70% and 75.81%, respectively.
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