人CD8+ T细胞的分离、激活与复苏技术解析

Isolation, Activation, and Recovery of Human CD8+ T Cells PBMCs were isolated from whole blood of healthy donors according to the described procedure. CD8+ T cells were then isolated from fresh human PBMCs using the STEMCELL Technologies EASYSEP Human CD8+ T Cell Isolation Kit. Briefly, PBMCs were mixed with the isolation antibody cocktail (provided in the kit) in PBS without Ca2+ and Mg2+, containing 2% FBS and 1 mM EDTA. After incubation at room temperature for 5 minutes, RapidSpheres™ magnetic beads (provided in the kit) were added, and the mixture was immediately placed on the STEMCELL Technologies EASY50 EASYSEP magnet. Following a 10-minute incubation at room temperature, the cell-containing supernatant was collected and placed back on the EASY50 EASYSEP magnet. After another 5-minute incubation at room temperature, the purified CD8+ T cells were collected and centrifuged. The purified CD8+ T cells were cryopreserved in liquid nitrogen for further use. To expand the CD8+ T cells, the purified human CD8+ T cells were resuspended in STEMCELL Technologies ImmunoCult™-XF T Cell Expansion Medium. The T cells were activated by adding STEMCELL Technologies ImmunoCult™ Human CD3/CD28 T Cell Activator and 100 U/mL human IL-2 from R&D Systems, and the cells were cultured for 12 days. During the 12-day expansion period, fresh ImmunoCult™-XF T Cell Expansion Medium containing 100 U/mL human IL-2 was added whenever the cell density exceeded 1 million cells per mL. At the end of the 12-day expansion, the expanded CD8+ T cells were cryopreserved in liquid nitrogen. To recover the CD8+ T cells, the expanded CD8+ T cells or cryopreserved CD8+ T cells were removed from liquid nitrogen and thawed in a 37°C water bath. The CD8+ T cells were resuspended in RPMI1640 medium containing 10% heat-inactivated FBS and 250 U/mL human IL-2. After a 48-hour incubation at 37°C, the CD8+ T cells were ready for further use.