For cell death analysis, cells were treated, collected, and initially stained with specific antibodies, then resuspended in PBS containing 1 μg/ml Propidium Iodide (PI) or 7-Aminoactinomycin D (7-AAD) for 5 minutes, and directly run on a flow cytometer. For cells expressing intracellular fluorescence proteins, cells were resuspended in PBS containing 1 μl LIVE/DEAD® Fixable Blue Dead Cell Stain (Thermo Fisher Scientific) for 20 minutes, then analyzed.
To quantify T cells and effector T cell cytokine expression, single-cell suspensions were prepared from fresh tumor tissues. T cells were enriched by density gradient centrifugation. For cytokine staining, T cells were incubated in culture medium containing PMA (5 ng/ml), Ionomycin (500 ng/ml), Brefeldin A (1: 1000) and Monensin (1: 1000) at 37°C for 4 hours. Anti-CD45 (30-F11), anti-CD90 (53–2.1), anti-CD4 (RM4–5), and anti-CD8 (53–6.7) were added for 20 minutes for surface staining. The cells were then washed and resuspended in 1 ml of freshly prepared Fix/Perm solution (BD Biosciences) at 4°C for overnight. After being washed with Perm/Wash buffer (BD Biosciences), the cells were stained with anti-TNFα (MP6-XT22) and anti-IFNγ (XMG1.2) for 30 minutes, washed, and fixed in 4% formaldehyde (Sigma Aldrich). All samples were read on an LSR II cytometer and analyzed with FACS DIVA software v. 8.0 (BD Biosciences).
To quantify T cells and effector T cell cytokine expression, single-cell suspensions were prepared from fresh tumor tissues. T cells were enriched by density gradient centrifugation. For cytokine staining, T cells were incubated in culture medium containing PMA (5 ng/ml), Ionomycin (500 ng/ml), Brefeldin A (1: 1000) and Monensin (1: 1000) at 37°C for 4 hours. Anti-CD45 (30-F11), anti-CD90 (53–2.1), anti-CD4 (RM4–5), and anti-CD8 (53–6.7) were added for 20 minutes for surface staining. The cells were then washed and resuspended in 1 ml of freshly prepared Fix/Perm solution (BD Biosciences) at 4°C for overnight. After being washed with Perm/Wash buffer (BD Biosciences), the cells were stained with anti-TNFα (MP6-XT22) and anti-IFNγ (XMG1.2) for 30 minutes, washed, and fixed in 4% formaldehyde (Sigma Aldrich). All samples were read on an LSR II cytometer and analyzed with FACS DIVA software v. 8.0 (BD Biosciences).