Annexin 5 binding buffer

To assess apoptosis, the cells were cultured for 24 h in RIPM 1640 medium containing normal glucose (2 g/L) or low glucose (40 mg/L) levels. The cells were centrifuged and resuspended in 0.5 ml annexin V-binding buffer (KeyGEN Biotech, China). Thereafter, 5 μl annexin V-APC and 7-AAD were added to the samples and incubated at RT for 10 min in the dark. The samples were then analyzed on a FACS Caliber flow cytometer (BD Biosciences, US).
The lymphocytes from 4T-1-injected BALB/c mice were isolated as follows: the tumor tissues from the mice were sectioned and digested with 2 mg/ml collagenase IV and 100 ng/ml DNase I (sigma) at 37 °C for 30 min. The tissues were then added to RPMI 1640 media supplemented with 10% FBS and 0.5 mM EDTA and separated by discontinuous 30–70% Percoll (GE Healthcare). After stimulation with PMA/Ionomycin and BFA (sigma) for 6 h at 37 °C, the cells were harvested for surface staining and intracellular staining (BD Pharmingen), according to the manufacturer’s instructions. The antibodies were anti-CD45-BV510, anti-CD45-APCcy7, anti-CD3-APC, anti-CD3-BV650, anti-CD4-FITC, anti-CD4-PEcy7, anti-PD-1-BV785, anti-PD-1-PE, anti-TIGIT-BV421, anti-TCR α/β-PE anti-IFN-γ-BV785, anti-IFN-γ-APC, anti-TNF-α-BV421 and anti-TNF-α-APC (BioLegend). All the data were collected by BD FACS Celesta flow cytometry and processed by Flow Jo software.