Anti pd 1 fitc

For staining of inhibitory receptors, stimulated PBMCs were surface stained with anti-CD4 (V500; BD Biosciences), anti-CD3 (APC-H7; BD Biosciences), anti-CD8 (Alexa Fluor 405; Invitrogen), anti-PD-1 (FITC; BD Bioscience) and anti-human TIM-3 antibody (PE; R&D Systems) for 30 min at 4°C. Cells were washed, fixed, permeabilized (Invitrogen), and intracellularly stained with anti-IFN-γ (PE-Cy7; BD Biosciences), anti-IL-2 (BV605; BD Biosciences), and anti-CTLA-4 (APC; BD Biosciences) for 30 min at 4°C, washed and fixed with 1% formaldehyde. Cells were analyzed using a LSR-II flow cytometer (BD Immunocytometry Systems). One million events were collected. Electronic compensation was performed with Ab capture beads (BD Biosciences) stained separately with individual monoclonal Abs used in the test samples. The data files were analyzed using Diva (BD) and FlowJo Software (Treestar, Co). The expression of TIM-3, PD-1 and CTLA-4 was examined on cytokine-producing cells with frequencies ≥0.03% above the background (media control tube) to ensure an adequate number of events for analysis as previously validated by our laboratory [15 (link)–18 (link)]. Flourescence minus one (FMO) controls were used to set the gates for determining the mean fluorescent intensity (MFI) of TIM-3, PD-1 and CTLA-4 on Gag-responsive T cells expressing IFN-γ or IL-2.

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.