Single cell suspensions were prepared directly from the cultured A549 and H1299 lung cancer cell lines or LLC tumor tissues and were then stained with the following conjugated, human specific monoclonal antibodies (BioLegend, San Diego, USA):
PE-PD-L2, PerCP/Cy5.5-Gal-9, PE/Cy7-CD200, and
PE/Cy7-PD-L1, and mouse monoclonal antibodies including
PE/Cy7-PD-L1,
PE/CD8,
FITC/CD4,
PE/Cy7-TIM3, and
APC-IFN-γ (BioLegend). The appropriate isotype controls purchased from BioLegend served as the negative controls. The cells were incubated with the indicated antibodies for 30 min at 4 °C when protected from light, washed twice with phosphate-buffered saline (PBS), and then resuspended in a final volume of 300 μL of 4% paraformaldehyde solution for analysis. All samples were analyzed in 3 independent experiments, and at least 20,000 events were acquired in each test.
Wang K., Wang J., Liu T., Yu W., Dong N., Zhang C., Xia W., Wei F., Yang L, & Ren X. (2021). Morphine-3-glucuronide upregulates PD-L1 expression via TLR4 and promotes the immune escape of non-small cell lung cancer. Cancer Biology & Medicine, 18(1), 155-171.
