Pe anti mouse pd l1

Cells were washed and stained with the following fluorescent antibodies: PE anti-human PD-L1 (#12-5983-42, eBioscience), PE anti-mouse PD-L1 (#124308, BioLegend) and APC anti-human MHC class I (#311409, BioLegend). After incubation, the cells were washed with PBS containing 0.5% BSA and run on a BD FACSVerse flow cytometer (BD Biosciences). A LIVE/DEAD™ Fixable Far Red Dead Cell Stain Kit (#L34974, Invitrogen) was used to gate live cells. The results were analyzed using FlowJo software (Tree Star, Ashland, OR, USA). For flow cytometric analysis of PD-L1 induced by IFN-γ, cells were treated with 10 ng/ml human IFN-γ (#PHC4031, Gibco) for 24 h.

To measure PD­L1 levels on the cell surface of tumour cells treated with vehicle, cisplatin, radiation, JQ1, IFN‐γ (20 ng/ml,²⁴ PeproTech), or the combinations for the different time periods were trypsinized and harvested for staining with streptavidin‐phycoerythrin (PE)‐conjugated anti‐human PD‐L1(329706, Biolegend). Tumour tissues were collected, cut into small pieces, digested with a mixture of collagenase, DNase and hyaluronidase for preparing single‐cell suspension and then treated with Cell Stimulation Cocktail (00‐4975‐03, eBioscience) as instructed. Cells were washed, re‐suspended in fluorescence‐activated cell sorting buffer at 4°C and then stained with fluorescent‐conjugated antibodies and appropriate isotype controls for multicolor flow cytometry. For cell surface staining, the following antibodies were used: fluorescein isothiocyanate (FITC) anti‐mouse CD3 (100204, Biolegend), PE/Cy7 anti‐mouse cluster of differentiation 4 (CD4) (100422, Biolegend), APC anti‐mouse CD8 (100712, Biolegend), Brilliant Violet 605 anti‐mouse NK1.1 (108739, Biolegend) and PE anti‐mouse PD‐L1(124308, Biolegend). The collected live single cells were fixed and treated with permeabilization buffer before intracellular staining with Brilliant Violet 421 anti‐mouse IFN‐γ (505830, Biolegend).