Anti pd 1 antibody

Single-cell suspension obtained and stained by following mAbs: Live/dead dye (Fixable Viability stain 620, BD Biosciences, 564,996), anti-CD45 antibody (BD Biosciences, 560,178), anti-CD3 antibody (BD Biosciences, 563,800), anti-CD4 antibody (BD Biosciences, 566,392), anti-CD8 antibody (BD Biosciences, 563,821), anti-CD25 antibody (BD Biosciences, 564,467), anti-CD127 antibody (BD Biosciences, 558,598), anti-CD45RA antibody (BD Biosciences, 563,031), anti-CCR7 antibody (BD Biosciences, 562,555), anti-PD-1 antibody (BD Biosciences, 563,245), anti-LAG3 antibody (BD Biosciences, 565,774), anti-CD39 antibody (BD Biosciences, 561,444). Single cell suspensions were stained with 1 μg/sample fluorochrome-labeled antibodies for specific surface marker at 4°C for 30 min in 100 μL PBS. Stained single cell suspension of tumor tissue were processed to flow cytometry using Cytoflex LX. The data were analysis by using FlowJo V10.6.2 software.

To determine the effect of 10 Gy radiation and various concentrations of IFN‐γ (5, 10, 20, 30 ng/mL) on PD‐L1 expression of 4T1, cells were harvested from each experimental group on days 3 and 5 after radiation or 24 hours after IFN‐γ stimulation. Then, anti‐PDL1 antibody (BD Biosciences, Franklin Lakes, NJ, USA) was used for flow cytometry analysis.
For PD‐1 detection, 4T1 cells pretreated with IFN‐γ (30 ng/mL) were collected and incubated with PBS or supernatant collected from 4T1/sPD‐1 cells media for 30 minutes. Anti‐PD1 antibody (BD Biosciences) was used for flow cytometry analysis.
On day 5 after vaccine or PBS injections, splenocytes and blood were isolated from mice of each group to count mature DC and PD‐1⁺ T‐cell subsets by staining cells with anti‐CD3, anti‐CD45, anti‐CD11c, anti‐CD83, anti‐CD86, anti‐CD8, anti‐CD4, and anti‐PD‐1 antibodies (BD Biosciences). Data acquired were processed with FlowJo version software (TreeStar, Ashland, OR, USA).