Pe conjugated anti pd l1

Flow cytometry was performed using a CytoFLEX S (Beckman Coulter Life Sciences, Mississauga, ON). Human LUAD cell lines A549, PC9, H1299, H1650, and H1975 were stained with 0.2 μg of PE-conjugated anti-PD-L1 (Biolegend, USA) antibody.

3D-O matrices were enzymatically digested with collagenase (20 mg/ml for 2–3 h at 37°C) on day 4. BCa cells were isolated and identified by gating cells with a high DiO signal (excitation, 488 nm; emission, 530/30 nm). Antibodies used to evaluate hypoxic status and surface marker expression were AlexaFluor 647 conjugated anti-HIF-1 α (359706, BioLegend, CA), APC-Cy7 conjugated anti-CD8 (344714, BioLegend, CA), PE-conjugated anti-PD-L1 (393608, BioLegend, CA), PE-conjugated anti-MUC-1 (355608, BioLegend, CA), and PerCP-Cy5 conjugated anti-CD73 (344014, BioLegend, CA). Cell viability was evaluated by using a Sytox Blue live-dead fluorescent dye (S34857, Invitrogen, CA) possessing excitation, 358 nm; emission, 461 nm or Live/Dead Blue cell stain (L34962, Thermo Fischer Scientific, MA). For all analyses, a minimum of 5,000 events were acquired using BD FACS Fortessa and FACSDiva v6.1.2 software or BD FACS Accuri and BS Accuri C6 software (BD Biosciences), respectively. The BCa cell counts were always normalized to a predetermined number of counting beads (424902, BioLegend, CA), and mean fluorescence intensity (MFI) ratios for each of the targets as mentioned above studied were assessed with respect to the corresponding isotype in the BCa-DiO⁺ cells. The data was analyzed using FlowJo program v10 (Ashland, OR).