Easysep direct human cd8 t cell isolation kit

Human CD8⁺ T cells were isolated and purified from healthy donor peripheral blood mononuclear cells (PBMCs) by an Easy-Sep™ Direct Human CD8⁺ T Cell Isolation Kit (STEMCELL Technologies). For CD8⁺ T cell activation and proliferation, human CD8⁺ T cells were seeded into 24-well plates, and anti-CD3/anti-CD28 antibodies (BD Biosciences) were added and incubated for 48 h. The miRNA mimic or scramble RNA was transfected into preactivated CD8⁺ T cells using a human T cell transfection kit (Lonza).

For the CD39⁺γδ Treg–mediated CD4⁺/CD8⁺ T cell suppression experiment, CD39⁺ γδ Tregs were sorted from tumors and paired normal tissues of patients with RSCRC/LSCRC and then were cocultured with allogeneic peripheral blood CFSE-labeled (5 μM; catalog 65-0850-84; Invitrogen) CD4⁺/CD8⁺ T cells at a 1:5 ratio in the presence of ImmunoCult Human CD3/CD28 T Cell Activator (catalog 10971; Stemcell Technologies) for 5 days. An anti-CD39 mAb (5 μg/mL; ab178572; Abcam) and an A2AR inhibitor, AZD4635 (5 μM; M7531; AbMole), were used for rescue experiments. CD4⁺/CD8⁺ T cells were separated by RosetteSep Human CD4⁺ T Cells Enrichment Cocktail (catalog 15062; Stemcell Technologies) and an EasySep Direct Human CD8⁺ T Cell Isolation Kit (catalog 19663; Stemcell Technologies). On day 5, cells were harvested and intracellularly stained with anti–IFN-γ mAb. CFSE-low cells and IFN-γ production were detected by flow cytometry. Peripheral blood samples of the healthy donor were collected in Tianjin Medical University Cancer Institute and Hospital.

The microporous 2-compartment co-culture system was applied for co-culture. Transfected NSCLC cells were inoculated in the upper chamber, while CD8⁺ T (PB009-3-C, ALLCELLS, Shanghai) cells were inoculated in the lower chamber, allowing direct contact between NSCLC cells and immune cells. CD8⁺ T cells were sorted using EasySep™ direct human CD8⁺ T cell isolation kit (STEMCELL, Vancouver, BC, Canada). Annexin V-FITC apoptosis kit (Becton Dickinson, Franklin Lakes, NJ, USA) was used for flow cytometry to determine the percentage of CD8⁺ T cells or apoptotic CD8⁺ T cells according to the manufacturer's instructions.

Fresh BM samples were obtained from patients at indicated times and BM mononuclear cell (BMMC) cells isolated after RBC lysis and density centrifugation. CD138⁺ cells were isolated from the BMMC fraction using the EasySep^™ human CD138 positive selection kit II (StemCell Technology, Cambridge, MA). CD8⁺ T-cells were isolated from the BMMC fraction using the EasySep direct human CD8⁺ T-cell isolation kit (StemCell Technology). The T-cell fraction was enriched from the BMMC fraction and free of CD138⁺ BM cells.

EasySep ™ Direct Human CD8+ T cell Isolation Kit (STEMCELL, Cambridge, MA, USA) was used to isolate CD8+ T cells from the whole blood of healthy individuals. Isolated cells were incubated with RPMI-1640 medium with 10% FBS.