Pe conjugated anti nk1

To determine the expression of PD-L1 by various immune cells and mature/immature BMM-derived DCs or M0/M1 BMM-derived macrophages, these cells were obtained, cultured, and harvested as described previously. To evaluate the effect of IFN-γ on PD-L1 expression of macrophages, BMM-derived M0 macrophages were treated with 500 U/mL recombinant murine IFN-γ (PeproTech) in a 5% CO₂ atmosphere at 37 °C for 24 h. To visualize surface PD-L1 expression on T cells, NK cells, NKT cells, MDSCs, and B cells, splenocytes were stained with FITC-conjugated anti-CD3 Ab (BioLegend), PE-conjugated anti-NK1.1 (BioLegend), PE-conjugated anti-CD11b (BioLegend), FITC-conjugated anti-Gr-1 (BioLegend), PE-conjugated anti-CD19 (BioLegend), and PerCP-eFluor 710-conjugated anti-CD274 (PD-L1) (eBioscience), respectively. To visualize the surface expression of PD-L1 on DCs and macrophages, BMM-derived DCs or BMM-derived macrophages were stained with PE-conjugated anti-CD11c Abor anti-F4/80 Ab (BioLegend), FITC-conjugated anti-PD-L1 Ab (BioLegend), and PE-Cy5-conjugated anti-CD80 Ab (BioLegend) or PE-Cy5-conjugated anti-CD86 or anti-MHC-II Ab (BioLegend), respectively. To visualize surface PD-L1 expression on TC-1 tumor cells, cells were stained with PE-conjugated anti-PD-L1 Ab (BioLegend). Flow cytometric analysis was performed using a FACSCalibur flow cytometer with CELLQuest software (BD Biosciences).