Cd135 pe

A panel of monoclonal antibodies was developed to enable phenotypic characterization of B and T lymphocyte subpopulations: CD3-FITC, CD4-APC, CD25-BV421, FoxP3-PE, CD19-PeCy7, CD9-BB700 (BD Bioscience, France), CD24-BV510, and CD38-APC-Cy7 (Sony Biotechnology, UK); hematopoietic stem and progenitor cells: CD34-BV421 (Biolegend, France), cKit-APC-H7, CD135-PE, lineage-FITC, Sca-1-BV510, CD16/32-PerCPCy5.5, CD127-APC (BD Bioscience), and TLR-4-PeCy7 (Biolegend); and dendritic cells: CD11c-PeCy7 (eBioscience, France), CMH-II-FITC, Siglec-H-BV510, CD11b-APC-H7, and CD103-PerCP5. For intracellular staining, cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences). For in vitro human B cell differentiation experiments, cells were stained with Fixable Viability Dye eFluor 450 to identify dead cells, followed by staining with CD25-BV605 (Biolegend), CD19-BUV395, CD9-FITC, CD27-BUV737, CD38-BV711 and intracellular IL-10-PE (BD Bioscience). Cells were analyzed on a Canto II flow cytometer (BD Biosciences). Data were acquired using Diva 8.0 software and analyzed with FlowJo X (TreeStar, Williamson Way, Ashland, USA). Fluorescence minus one staining controls were used for all panels, and dead cells were removed using viability staining.

Six weeks after tumour cell injection, mice were euthanized humanely. The femurs, tibias, and iliac crest were harvested immediately and placed in PBS. Bone marrow cells were obtained by rinsing femurs, tibias, and iliac crest with PBS. After collection, 1 × 10⁶–5 × 10⁶ cells were suspended in 100 μl of FACS buffer and stained with the appropriate antibodies at 4 °C. According to the manufacturer’s protocol, 10 μl of each antibody was sequentially added to the cell suspension on ice. The workflow is shown in Supplementary Figure 1. Fresh BM cells were stained with the following antibodies: lineage antibodies (B220 (RA3-6B2), CD3 (17A2), Gr-1 (RB6-8C5), CD11b (M1/70), CD8a (53–6.7), CD4 (GK1.5) and Ter-119 (TER-119)), Sca-1-Pe-Cy7 (D7, BD Biosciences), c-Kit-APC (2B8), CD150-PE (TC15-12F12.2), CD48-PerCP (HM48-1, eBioscience), CD41-FITC (MWReg30), CD34-FITC (RAM34, eBioscience), CD16/32-BV421 (93), CD71-FITC (C2, BD Biosciences), CD135-PE (A2F10) and CD138-PE (281-2).