Surface flow cytometry analysis of PD-L1 and MHC class I

On day 1, murine cancer cell lines (B16, MC38 and YUMMER2.1) were plated at 2×10⁵ per 6-well plate and, when cell confluence reached 70–80%, cells were collected for surface staining. The day after, culture media was replaced with fresh media with or without IFNγ 100 ng/ml (PeproTech, Cat #315-015) for 18 hours. On day 3 after incubation, cells were trypsinized and then incubated at 37°C for 2 hours with the same concentrations of IFNγ. Next, cells were centrifuged to remove the media and resuspended in 100% FBS. Cells were first stained with Zombie Green viability dye (BioLegend, San Diego, CA Cat #423111) for 15 minutes, then washed and stained with PE-conjugated anti-mouse PD-L1 (clone MIH5, BD Biosciences, Cat #558091), APC-conjugated anti-mouse MHC I (clone 28-14-8, eBioscience, Cat #17-5999-82) and AF700-conjugated anti-mouse MHC II (clone M5, eBioscience, Cat #56-5321-82), and left on ice for 20 minutes. Cells were then washed once with 3 ml PBS and resuspended in 300 μL of PBS. Following staining, samples were analyzed using the Attune Flow Cytometer (Thermo Fisher Scientific) platform at the UCLA JCCC Flow Cytometry Core. Data were analyzed using FlowJo software (version 10.0.8r1, Tree Star Inc., San Carlos, CA). Experiments were performed at least in duplicate per cell line.