Cd4 v450 clone rpa t4

Antibodies used for flow cytometry include: pan-γδTCR-PE (clone IMMU510; Beckman Coulter, Woerden, the Netherlands), pan-αβTCR-APC (clone IP26; eBioscience, Thermo Fisher Scientific), CD4-V450 (clone RPA-T4; BD Biosciences), CD8α-PerCP-Cy5.5 (RPA-T8; Biolegend), CD3-eFluor 450 (OKT-3; eBioscience), CD45-FITC (2D1; BD Biosciences), CD16-FITC (3G8; BD Biosciences), CD56-FITC (MY31; BD Biosciences), CD27-APC-eFluor780 (O323; eBioscience), CD45RO-PE-Cy7 (UCHL-1; BD Biosciences). All samples were analyzed on a BD LSRFortessa using FACSdiva software (BD Biosciences).

Cell phenotype, activation state, and percentage of monocyte subsets were determined by 8-color flow cytometry using a FACSCanto II (BD Biosciences, San Jose, CA, USA). Differential gating was based on size and granularity (Figure 1a,b), with monocyte lineage defined as CD4^+loD11c⁺CD14⁺CD16^+/−, classical monocytes as CD14^hi++CD16⁻, intermediate monocytes as CD14⁺⁺CD16⁺, and non-classical monocytes as CD14^+loCD16⁺⁺ (Figure 1d) [21 (link),22 (link)].
Cells were stained with monoclonal antibodies reactive with CD14^V500 (clone M5E2), CD274/PD-L1^PE (clone MIH1), CD25^PE−Cy7 (M-A251), CD16 ^APC−Cy7 (clone 3G8), CD4^V450 (clone RPA-T4) (BD Biosciences), and CD11c^PerCPCy5.5 (clone 3.9, BioLegend, San Diego, CA, USA). Fluorescence minus one (FMO) controls were used to set gates for CD25 and programmed death-ligand 1 (PD-L1). Unstimulated samples were used to set gates for intracellular cytokines.