Cd155 pe

De-identified surgically resected tumor tissue was collected from consented subjects under an IRB-approved protocol. GBM tumor tissue was collected within 1h of resection by the Duke Preston Robert Tisch Brain Tumor Center BioRepository. Specimens were dissociated in RPMI-1640 containing 100μg/mL Liberase-TM (Sigma-Aldrich) and 10μg/mL DNAse I (Roche) for 20min at 37°C with agitation. Single cell suspensions were filtered through 70μM and 40μM cell strainers (Olympus Plastics), washed in PBS (Gibco), and reconstituted in PBS containing 2% FBS (Sigma-Aldrich) with 1:20 Human Tru-Stain FcX™ block (Biolegend). Cell suspensions were stained with antibodies against CD45-BUV395 (BD Biosciences), CD14-BV421, CD33-BV510, HLA-DR-BV786, CD31-FITC, CD3/19-BUV737, CD11b-APC, CD16-BV711, CD15-APC-fire7, and either CD155-PE and PD-L1-BV605 or isotype control-PE and -BV605 antibody (all BioLegend); followed by washing and reconstitution in 7-AAD containing PBS+2%FBS. Cells were gated for appropriate size on SSC-A and FSC-A; single cells by proportionate FSC-H and FSC-A size; live cells by 7-AAD^Neg; non-endothelial cells by CD31-FITC-A^Neg; CD45 by CD45-BUV396-A⁺, non NK, B, or T Cells by CD56-BUV737-A^-, CD19-BUV737-A^-, and CD3-BUV737-A^-; and for macrophages by CD11b-APC-A⁺, CD16-BV711-A⁺,and CD14-BV421-A⁺.