Pe anti mouse pd l1

FITC mouse IgG1 κ isotype control (400110), PerCP-Cy5.5 mouse IgG1 κ isotype control (400149), PE mouse IgG1 κ isotype control (400114), PerCP-Cy5.5 anti-human CD3 (300430), FITC anti-human CD8a (300906), PE anti-human CD45 (368510), PerCP-Cy5.5 anti-human IFN-γ (506528), FITC Rat IgG2a κ isotype control (400506), PerCP-Cy5.5 Rat IgG1 κ isotype control (400426), FITC anti-mouse CD3 (100204), FITC anti-mouse CD8a (100705), PE anti-mouse CD45 (103106), and PerCP-Cy5.5 anti-mouse IFN-γ (505822) were purchased from Biolegend. PE anti-human PD-L1 (557924), FITC anti-human PD-L1 (558065), FITC anti-human HLA-A2 (343303), FITC Rat IgG2b κ isotype control (400605), PE Rat IgG2a λ isotype control (400635), and PE anti-mouse PD-L1 (558091) were obtained from BD Biosciences.

For isolation of peripheral lymphocytes, 500 μl peripheral blood were collected from each mouse and lysed by treatment with 1 ml lysing Solution (BD Biosciences, Franklin Lakes, NJ, USA) to clear red blood cells. The obtained lymphocytes were incubated with fluorescence-conjugated monoclonal antibodies in staining buffer. Eight-color flow cytometry analysis (n = 8) was performed on a FACSCalibur device (Becton Dickinson, Mountain View, CA, USA). The frequency of positive cells was analyzed using the program Cell Quest in two regions. The lymphocyte region was determined using granularity (SSC) and size (FSC) plots. Tfh cells were identified as a CD4⁺lineage⁺ (CXCR5⁺, ICOS⁺, FOXP3⁺, IL-10⁺, IL-17⁺, and BCL-6⁺) population, and within this group, the CD4⁺ population was assessed. The following monoclonal antibodies were used: APC-H7 anti-mouse CD4 (No. 560181) (1:200), FITC Anti-Mouse CXCR5 (No. 560577) (1:100), PE Anti-Mouse ICOS (No. 565669) (1:100), PerCP-Cy5.5 Rat Anti-Mouse FOXP3 (No. 563902) (1:100), APC Anti-Mouse IL-10 (No. 554468) (1:200), APC-Cy7 Anti-Mouse IL-17 (No. 560821) (1:200), APC Anti-Mouse PD-1 (No. 562671) (1:200), PE Anti-Mouse PD-L1 (No. 558091) (1:200), and PerCP-Cy5.5 Anti-Mouse BCL-6 (No. 563582) (1:200) (BD Biosciences, Franklin Lakes, NJ, USA). Limits for the quadrant markers were always set based on negative populations and isotype controls.