Cd8 pe cy7 clone rpa t8

Flow cytometry was performed on PBMCs from baseline, after Cycle 1, after Cycle 3, and after surgery using an antibody panel designed to identify tumor-related T cells, Effector cytotoxic T lymphocytes (CTLs), and pro-apoptotic T cells. To perform flow cytometry analysis of the phenotype and functional potential of immune cells, the following antibodies were used as per published protocols^{43 (link),44 (link)}: CD8-PE-Cy7 (clone RPA-T8, catalog 304006, BD Pharmingen), CD11a-APC (clone HI111, catalog 301212, BioLegend), PD-1 fluorescein isothiocyanate (FITC) (clone EH12.2H7, catalog 32990, BioLegend), CX3CR1-APC/Cy7 (clone 2A9-1, catalog 341616, BioLegend), Bim-PE (clone C34C5, catalog 12186S, Cell Signaling Technology), NKG7 monoclonal antibody (AG1490 Rb mAb 8H3/8K3)-FITC conjugated (Fusion antibodies with a contract with Dong lab). T cells were stained for surface markers before intracellular staining. Data was collected on a CytoFLEX LX (Beckman Coulter, Atlanta, GA) and analysis was performed with the R software version 4.1.1.

Whole blood was stained with LIVE/DEAD fixable violet stain (ThermoFisher Scientific, Montigny le Bretonneux, France), CD3-PerCP (clone SP34-2, BD Biosciences, Le Pont-de-Claix, France), CD20-PE (clone 2H7, BD Biosciences, Le Pont-de-Claix, France), HLA-DR-ECD (clone immu357, Beckman Coulter, Villepinte, France), CD8-PECy7 (clone RPA-T8, BD Biosciences, France), CD14-APC (clone TÜK4, Miltenyi Biotec, Paris, France) and CD16-APC-AF750 (clone 3G8, Beckman Coulter, Villepinte, France). Monocytes and PMNs were analyzed in whole blood after fixation with paraformaldehyde (1%). Cells were analyzed using an Attune NxT acoustic focusing cytometer (ThermoFischer Scientific, Montigny le Bretonneux, France) with a violet scatter filter (NxT No-Wash No-Lyse filter) installed with a collection rate of 200 µL/min. Analyses of two time points (4 and 24 h) were performed at the same time in order to attain comparative analyses of fluorescence intensities.