Easysep mouse cd8 t cell isolation kit

OT1CD8⁺ T cells were isolated from the spleen of OT1 mice (Cavens Biogle, Suzhou, China) using EasySepTM Mouse CD8+ T Cell Isolation Kit (#19853, STEMCELL Technologies), following the manufacturer's instructions. OT1CD8⁺ T cells were maintained in RPMI 1640 supplemented with 5% FBS, 55 µm β‐mercaptoethanol, 1% penicillin/streptomycin, and 10 ng mL⁻¹ IL‐2. OT1CD8⁺ T cells were stimulated with 2 µg mL⁻¹ SIINFEKL and 10 ng mL⁻¹ IL‐2 for 3 days to generate CTLs. CTLs were cocultured with mCherry‐expressing MB49‐OVA cells for 24 h at a ratio of 2:1, with or without MK‐2206 (S1078, Selleck) or EGCG (S2250, Selleck). Cytotoxicity was assessed by calculating the percentage of 7‐AAD+ cells among mCherry+ cells using flow cytometry.

EasySepTM Mouse CD8⁺ T‐cell Isolation Kit (cat#19853, Stemcell Technology) was used to isolate CD8⁺ T cells. First, mouse spleen was isolated from 8‐weeks‐old C57BL/6 mice and passed it through a 70‐μm cell strainer by syringe to produce single‐cell suspensions. After removing red blood cells, EasySepTM Mouse CD8⁺ T cell Isolation Kit was used to separate CD8⁺ T cell from single‐cell suspensions according to the manufacturer's instructions. CD8⁺ T cells were previously cultured in 1640 medium containing 10% FBS, 2.5 μg/mL anti‐CD3 (cat#16‐0031‐86, ebioscience) and 5 μg/mL anti‐CD28 (cat#16‐0281‐86, ebioscience) for 24 hours before cocultured with luciferase‐labeled Hepa1‐6 cells. 5000/well luciferase‐labeled Hepa1‐6 cells were planted into 96‐well culture plates and treated with 5 µmol/L Oxysophocarpine for 24 hours before cocultured with CD8⁺ T cells. Then, CD8⁺ T cells co‐cultured with Oxysophocarpine‐treated Hepa1‐6 cells with the ratio of 5:1 and 10 μg/mL Lag‐3, PD‐1, TIGIT, or Tim‐3 antibody (cat#BE0174, BE0146, BE0274, BE0115, BioXcell, USA) was added into coculture system for 48 hours at 37℃. To determine the lysis level of Hepa1‐6 cells, D‐luciferin was added into the 96‐well plates. Fluorescence microplate reader was used to detect the fluorescence values of each well.

CD8 cytotoxicity assays were performed as previously described.^{91 (link)} Briefly, CD8 T cells from TRP1^high Mice^{57 (link)} were isolated with EasySep Mouse CD8+ T cell Isolation Kit (StemCell), resuspended in complete RPMI +100U/mL hIL2 (Peprotech #200–02) and stimulated with CD3/CD28 beads Dynabeads (Thermofisher #11456D) for 48 h. Target B16^Nectin1 or ex vivo cultures were plated in complete DMEM with 50 ng/mL IFNγ (Peprotech #315–05) on the day before co-culture. On day seven following T cell isolation, CD8 T cells were plated at various E:T ratios with or without the presence of tumor cells. 24 h following co-incubation of T cells with tumor cells, media was carefully removed and cell viability was assessed using CellTiterGlo (Promega). Relative luminescence was normalized to wells containing the same number of T cells without tumor cells present. For T cell cytotoxicity comparing wildtype B16^Necti1 to β2m^−/− B16^Nectin1, tumor cells were stained with 5μM CFSE prior to plating with 50 ng/mL IFNγ; T cell cytotoxicity was measured as fluorescent confluence loss comparing T cell containing wells to wells with tumor cells only using a Celigo image cytometer (Nexcelom 200-BFFL-5c).

Spleens from the tumor-bearing C57BL/6 mice or cryo-thermal treated mice were harvested and splenocytes were prepared using GentleMACS™ dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany) and passed through a 40-μm nylon filter. CD4⁺ T cells were isolated by EasySep™ Mouse CD4 Positive Selection Kit II (StemCell Technologies, Vancouver, BC, Canada). CD8⁺ T cells were isolated by EasySep™ Mouse CD8⁺ T Cell Isolation Kit (StemCell Technologies, Vancouver, BC, Canada). Natural Killer (NK) cells were isolated by EasySep™ Mouse NK Cell Isolation Kit (StemCell Technologies, Vancouver, BC, Canada). CD68⁺ macrophages were isolated by EasySepTM PE positive selection kit (StemCell Technologies, Vancouver, BC, Canada) and CD68-PE (clone FA-11, Biolegend, San Diego, CA, USA). DCs were isolated by EasySep™ Mouse Pan-DC Enrichment Kit II (StemCell Technologies, Vancouver, BC, Canada). Cells were all isolated according to the manufacturer’s instructions. Cells with a purity of >90% were used for experiments.

Cytotoxic responses were evaluated according to the manufacturer’s instructions (Promega, Madison, WI, USA). Briefly, splenic CD8⁺ T cells were isolated from mice immunized with PBS or with 10 μg LEX-CD80, LEX-CD86, LEX-CD8086 or LEXnull. Seven days after the last stimulation, splenic CD8⁺ T cells were isolated from immunized mice using an EasySep™ mouse CD8⁺ T cell Isolation Kit (Stem-cell Technologies, Vancouver, Canada). Cells were re-stimulated with irradiated L1210 cells for 7 days and then harvested as effector cells. L1210 cells were used as specific target cells and p388 cells were used as controls. The cells were seeded in a 96-well plate at 1 × 10⁴ cells/well. After adding CytoTox 96^® Reagent (Promega, Madison, WI, USA), absorbance at 490 nm was recorded and cells were incubated for 30 min, protected from light. The magnitude of the cytotoxic response at different effector/target (E/T) ratios was evaluated using a LDH assay (Promega, Madison, WI, USA), and specific lysis (%) was calculated as follows: (experimental LDH release − effector cells − target spontaneous LDH release)/(target maximum LDH release) × 100 [28 (link)].