Vista bv421

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).