Pe conjugated anti mouse pd l1 antibody

For cell cycle analysis, maltol- or cisplatin-treated B16F10 cells were fixed in ice-cold 70% ethanol overnight at 4°C. Then, the cells were incubated in PBS buffer containing RNase (100 μg/mL) and propidium iodide (PI, 50 μg/mL) for 30 min at room temperature, utilizing a PI flow cytometry kit (abcam, Cambridge, United Kingdom). For apoptosis analysis, B16F10 cells treated with maltol or cisplatin were stained with annexin V and PI using a FITC annexin V apoptosis detection kit with PI (Biolegend, San Diego, CA, United States). Cell surface PD-L1 detection was performed with reference to previous protocols (Tang et al., 2018 (link); Xu et al., 2018 (link)). Maltol or cisplatin-treated B16F10 cells were first blocked with anti-CD16/32 (anti-FcγIII/II receptor, clone 2.4G2, BD Biosciences, ≤ 1 µg/million cells), then incubated with PE-conjugated anti-mouse PD-L1 antibody (12-5982-83, eBioscience, San Diego, CA, United States, 1:200) in FACS buffer (PBS containing 0.1% BSA and 0.02% NaN₃) for 30 min at 4°C. Following PBS washes, the samples were resuspended in FACS buffer and analyzed through flow cytometry.

KPC cells pretreated with shRNAs were incubated with IFNγ (50 ng mL^− 1) for 48 h. Cells were then detached from culture dishes using 4 mM EDTA in PBS, collected by centrifugation, and incubated with either PE-conjugated anti-mouse PD-L1 antibody (Clone: MIH5, eBioscience) or an isotype-matched antibody in PBS with 2% FCS for 40 min on ice. After washing with PBS, cells were then incubated with 7AAD (BD Pharmingen) in PBS for 10 min on ice, and subjected to flow cytometric analysis (BD FACSVerse™).