Pd 1 pe antibody

After red blood cell lysis, WBCs were collected from 2 mL of the patients’ blood and subjected to flow cytometry after surface staining using a CD4-FITC antibody (eBiosciences; San Diego, CA, USA), a CD8a-PE-Cy7 antibody (eBiosciences), and a PD-1-PE antibody (BD Pharmingen, San Jose, CA, USA). A total of 2 × 10⁵ WBCs were resuspended in 100 μL of RPMI medium with 10% FBS, 0.25 µg of the CD4-FITC antibody, 0.06 µg of the CD8a-PE-Cy7 antibody, and 20 µL of the PD-1-PE antibody. The corresponding isotype control antibodies were used under the same conditions. After staining for 1 h, the cells were washed using 2 mL of phosphate-buffered saline solution containing 10% FBS and 1 mM EDTA. The fluorescence measurements were performed using a CytoFLEX Flow cytometer (Beckman Coulter, Brea, CA, USA). The gating strategies were (1) 2 × 10⁴ events for CD4⁺/CD8⁺-gated cells, (2) positive and negative signals for PD-1 expression were based on the CD4⁺ or CD8⁺ cursors to yield positive signals after subtracting the signal from the PE-conjugated isotype control, and (3) the percentages of CD4⁺ and CD8⁺ cells were calculated, as well as the proportions of CD4⁺ and CD8⁺ cells that were positive for PD-1.

Flow cytometry was performed as previously described.^{24 (link)} in brief, HN4 and HN30 cells were incubated with the indicated agent for 48 h. The cells were collected and incubated with anti-human PDL1 antibody at 1:100 (BD Biosciences, Franklin Lakes, NJ) for 30 min on ice. Then, the cells were resuspended in 100 μl fluorescence-activated cell sorting buffer and analysed on BD Fortessa flow cytometer. The final results were analysed with FlowJo software. Signal intensity was calculated as the ratio of the median fluorescence of the PDL1 antibody to that of the isotype control antibody (SFI: specific fluorescence index). CD4-FITC antibody, CD8-PerCP-Cy5.5 antibody, CD56-APC antibody, and PD1-PE antibody (all purchased from BD Biosciences) were applied to detect the PD1 expression on the surface of immune cells from peripheral blood of HNSCC patients and healthy controls. The IFNAR1 antibody (Abcam, Cambridge, MA, UK) and PE-conjugated secondary antibody (Proteintech, Rosemont, IL, USA) were used to analyse the surface IFNAR1 expression on immune cells.