Anti cd16 32 pe cy7

BM-MNC (1.5 × 10⁶ cells/sample) were stained with appropriate conjugated antibodies in PBS with 2% BSA. Specifically, human BM-MNC were stained with anti-CD33-PE-Cy7, anti-CD34-PerCP-Cy5.5, anti-CD14-BV510, anti-CD71-BV421, anti-CD38-BV711, anti-CD235a-BUV395, anti-PD-1-FITC, and anti-PD-L1-APC (BD Biosciences). Gating strategies for the identification of myeloid cells, HSPCs, and erythroid progenitors are shown in Supplementary Figure S1. Mouse BM cells were stained with anti-c-Kit-PerCP-Cy5.5, anti-Sca-1-PE, Lin-APC (including anti-CD3ε, anti-CD11b, anti-CD45R/B220, anti-TER-119, anti-Gr-1), anti-PD-1-BV421, anti-PD-L1-BV711, anti-PD-L2-BUV395, and anti-CD16/32-PE-Cy7 (BD Biosciences). Cell viability was determined using near-infrared live/dead dye (BD Biosciences) and the negative (live cell) population was used for further analysis. Samples were acquired on an LSR II flow cytometer and analyzed using FlowJo 9.9.3 software. Intracellular staining with PE-conjugated anti-active caspase-3 was performed using the Cytofix/Cytoperm™ protocol following initial cell surface receptor staining (BD Biosciences). For PD-1/PD-L1 ligation experiments, 2 million BM-MNC were plated per well in 24-well plates coated with recombinant human PD-L1 (2 μg/mL) for 24 h at 4 °C.