Facs flow cytometer

PE Annexin V Apoptosis detection kit (BD Biosciences, CA) was used to detect apoptosis. Cells were treated with cisplatin (12 μM) or carboplatin (80 μg/ml) for 48 h and were collected by centrifugation, resuspended in 400 μl binding buffer, followed by staining with 5 μl PE Annexin V and 5 μl 7-ADD for 15 min in dark at room temperature. Apoptotic cells were then evaluated by 7-ADD and Annexin V-positive cells on a fluorescence-activated cell-sorting (FACS) flow cytometer (Beckman Coulter, CA).

The cell proliferation assay was evaluated using a Cell Counting Kit‐8 assay (CCK‐8; Dojindo, Gaithersburg, MD, USA) according to the manufacturer's protocol. Briefly, 3 × 10³ cells/well incubated in 100 μl culture medium in 96‐well plates and treated with 10 μl/well of CCK‐8 solution during the last 4 hr of culture. The absorbance at 450 nm was measured in a ThermoMax Microplate Reader (Molecular Devices, Sunnyvale, CA, USA).
For cell cycle analysis, after transfection for 48 hrs, cells were harvested and fixed with ethanol overnight, then stained with propidium iodide (PI; Calbiochem, San Diego, CA, USA), and examined with a fluorescence‐activated cell sorting (FACS) flow cytometer (Beckman Coulter, Pasadena, CA, USA).

The single-cell suspensions were generated in PBS buffer with 1% FBS and 0.1 mM EDTA. Cells were incubated with APC-conjugated anti-human PD-1 antibody (Clone EH12.2H7, Biolegend) for 30 min at 4 °C in the dark, then the cells were washed twice with PBS buffer containing 1% FBS and 0.1 mM EDTA. Flow cytometry data were acquired on a FACS flow cytometer (Beckman, Cytoflex) and analyzed with Flowjo 10.6.2 software (TreeStar).

The harvested spleen was minced gently with the plunger of a 10 mL syringe and passed through a 70-μm nylon mesh cell strainer (Falcon, New Jersey, USA) to achieve a single cell suspension. Red blood cells were removed by cell lysis buffer (BD Biosciences, San Jose, USA). For intracellular cytokine staining, harvested cells were stimulated with phorbol myristate acetate (PMA), ionomycin, and brefeldin A (Cell Activation Cocktail, Biolegend, San Diego, USA) for 5 h. FITC-anti-CD4 (RM4-5), APC/Cy7-anti-CD8a (53-6.7), and matched isotype control antibodies were purchased from BD Biosciences. PerCP/Cy5.5-anti-CD4 (RM4-5), FITC-anti-IFN-γ (XMG1.2), APC-anti- interleukin (IL)-4 (11B11), and matched isotype control antibodies were purchased from Biolegend. Flow cytometric analysis was performed using a FACS flow cytometer (Beckman Coulter, Miami, USA), and analyzed by Kaluza software (version: 1.1; Beckman Coulter).

Apoptosis analysis was conducted with an Annexin V-FITC Apoptosis Detection Kit (KeyGen Biotech) according to the manufacturer’s protocol. The percentage of apoptotic cells was determined using FACS flow cytometer equipped software (BECKMAN).