Mhc 2 bv650

Blood obtained from cardiac bleeds was used to profile circulating immune cells after red blood cell lysis (155 mM NH4Cl, 10 mM KHCO₃, 0.1 mM EDTA, pH 7.3). Cells were stained with the following antibodies: CD8a-PE-Cy7 (53-6.7), CD4-APC-Cy7 (GK1.5), CD69-APC (H1.2F3), CD279-PE (J43), Ly6G-BV421 (1A8), B220 Pe-Cy5, CD103 bv510, CD11b BV605, CD11c Percpm, CD206 APC, CD25 APC, CD27 A-Cy7, CD4 A-Cy7, CD4 Pe-Cy7, CD44 FITC, CD62L BV450, CD69 FITC, CD69 Pe-Cy7, CD8 A-Cy7, CD8 APC, CD8 Pe-Cy5, CD8 Pe-Cy7, CD80 PE, F4/80 Pe-Cy7, FOXP3 FITC, H2-Kd FITC, IFNAR1 PE, IFNg PE, Ly6C-APC, Ly6G BV711, MHC-II BV650, NKG2D Pe-Cy7, NKp46 BV421, PD1 PE, PDL1 BV421, Rat IgG2a FITC, TCRbeta BV510, TNFa FITC (all from BD Biosciences) and Ly6C-APC (HK1.4) (Biolegend). Primary tumors were mechanically and enzymatically digested with 1 mg/mL collagenase I (Sigma) and 30 μg/mL DNAse I (Sigma) at 37 °C to obtain a single cell suspension before red blood cell lysis. Analysis of tumor-infiltrating lymphocytes was done as above. Analysis of immune cell populations was performed by flow cytometry using a FACSCanto II (BD Biosciences, USA). Data was analyzed using Flowjo software (TreeStar, USA).

PBLs and cancer cells from direct and indirect co‐cultures were used for multi‐marker flow cytometry to detect the expression of immune checkpoint markers. Cells were washed and resuspended in 100 μl of staining buffer containing Human Fc Block™ (564219, BD Biosciences). The expression of immune checkpoint receptors was determined using the following antibodies: PD‐1 PE‐Dazzle 594 (329940, Biolegend), VISTA BV421 (566750, BD Biosciences), CTLA‐4 BV786 (563931, BD Biosciences), TIM‐3 BV650 (565565, BD Biosciences), LAG‐3 PE (565617, BD Biosciences), TIGIT BUV395 (747845, BD Biosciences) and CD8 BV510 (563919, BD Biosciences). In parallel, we determined the expression of respective ligands: CD80 BV510 (740150, BD Biosciences), CD86 Alexa700 (564544, BD Biosciences), PD‐L1 PE‐Cy7 (558017, BD Biosciences), PD‐L2 BV786 (563843, BD Biosciences), VISTA BV421 (566750, BD Biosciences), MHC‐II BV650 (564231, BD Biosciences), GAL‐9 PE (565890, BD Biosciences) and PVR BUV395 (748272, BD Biosciences). Flow cytometry was performed by recording 50 000 events/sample using the LSRFortessa X‐20 instrument and FlowJo V10.7.1 software (BD Biosciences).