Apc conjugated anti cd19

PE-conjugated anti-CD23 (eBioscience-B3B4), FITC-conjugated anti-IgM (Sigma-μ chain specific) or brilliant violet (BV) 421-conjugated anti-IgM (BD pharmingen-R6-60.2), APC-conjugated anti-CD19 (BD bioscience-1D3), FITC-conjugated anti-CD3ε (Immunotools-145-2C11), APC-conjugated anti-CD11b (Biolegend- M1/70) and PE-conjugated anti-Gr-1(Immunotools- RB6-8C5) were used for flow cytometric analysis of blood leukocytes. Biotinylated anti-B220 (eBioscience-RA3-6B2) or Alexa 647-conjugated anti-B220 (BD Pharmingen- RA3-6B2), PE-conjugated anti-IgD (Biolegend-clone 11-26c.2a), FITC-conjugated anti-IgM (Sigma-μ chain specific) or BV 421-conjugated anti-IgM (BD Pharmingen- R6-60.2), FITC- conjugated anti-CD21 (BD Pharmingen- eBio8D9), PE-conjugated anti-CD23 (eBioscience-B3B4), APC-conjugated anti-CD19 (BD bioscience-1D3) or Alexa 700-conjugated anti-CD19 (BD Pharmingen-1D3) were used for flow cytometric analysis of BM or spleen cells. Biotinylated antibodies were detected with APC-conjugated streptavidin (Immunotools) or BV 605-conjugated streptavidin (BioLegend). Rat anti-mouse SIRPα (mAb P84; rat IgG1; a generous gift from Dr.Carl Lagenaur, university of Pittsburgh) was purified and conjugated to Alexa 488 as previously described [18 (link)].

Cells were sorted by Fluorescent-Activated Cell Sorting (FACS) into three cell populations: CD4⁺ (T cells), CD14⁺ (monocytes), and CD19⁺ (B Cells). Human PBMCs were stained with the following antibodies from BD Biosciences (San Jose, CA, USA): FITC conjugated anti-CD14 (347439#), APC conjugated anti-CD19 (340437#), and PE conjugated anti-CD4 (347327#). All samples were treated with a LIVE/DEAD cell viability assay (Invitrogen, Carlsbad, CA, USA) and gated on cell viability. Two samples with less than 70% cell viability were excluded (n = 18). Lymphocytes and monocytes were sorted directly into 24-well plates with 0.75 mL Trizol Reagent (Invitrogen, Carlsbad, CA, USA) to maximize RNA extraction.

For phenotypic analysis, single-cell suspensions were prepared from spleen, lymph nodes, liver and thymus. Briefly, lymph nodes and thymus were passed through a 200-gauge steel mesh and washed with PBS. Spleens were firstly passed through a 200-gauge steel mesh and then RBC were lysed and remaining cells washed with PBS. Livers were passed through a 200-gauge steel mesh and the cell pellets were collected in the centrifuge tube, the mononuclear cells in the pellets were isolated by gradient centrifugation with 40% and 70% Percoll. Single-cell suspensions were stained according to the manufacturers’ instructions using fluorochrome-coupled monoclonal antibodies (mAbs). The following mAbs were obtained from BD Bioscience: BV421-conjugated anti-CD45.2 (Catalog # 562895), PE-conjugated anti-CD5 (Catalog # 553022), APC-conjugated anti-CD19 (Catalog # 550992). PE-Cy7-conjugated anti-IL-10 (Catalog # 505026) was purchased from Biolegend. Flow-cytometric analyses were performed using an LSR II flow cytometer (BD Biosciences) and flow-cytometry data were analyzed using Flowjo V10 software (BD Biosciences).