Igg1 mopc 21

TLR9 (J15A7; BD Biosciences; San Jose, CA), IgG1 (MOPC-21; BD Biosciences; San Jose, CA), CD45 (30-F11; BD Biosciences; San Jose, CA), CD11b (M1/70; BD Biosciences; San Jose, CA), CD11c (N418; Biolegend; San Diego, CA), Siglec F (E50-2440; BD Biosciences; San Jose, CA), MHC II (I-A/I-E) (M5/114.15.2; BD Biosciences; San Jose, CA), CD64 (X54-5/7.1; Biolegend; San Diego, CA), F4/80 (BM8 eBiosciences; SanDiego, CA), LY6G (1A8; BD Biosciences; San Jose, CA), CD3 (17A2, BD Biosciences; San Jose, CA), CD90.2 (53–2.1; BD Biosciences; San Jose, CA), CD4 (GK1.5; Biolegend; San Diego, CA) CD8 (53–6.7; BD Biosciences; San Jose, CA), NKP46 (29A1.4; Biolegend; San Diego, CA), CD19 (1D3; BD Biosciences; San Jose, CA), Fc Block(CD16/CD32) (2.4G2; BD Biosciences; San Jose, CA). The NP antibody used was MA1-7322 from Thermofisher (Waltham, MA) conjugated to FITC.

Whole blood cells were stained with antibodies, as described previously [9 (link),11 (link)]. Briefly, whole blood was aliquoted into microtubes (100 μL of blood per tube) and incubated with an Fc Blocker (BioLegend, San Diego, CA, USA) for 5 min. After blocking the Fc receptors, the whole blood was incubated with each antibody for 30 min at 4 °C and then treated with pre-warmed BD Phosflow Lyse/Fix buffer (1 mL; BD Biosciences, San Jose, CA, USA) for 10 min at 37 °C to lyse the red blood cells and fix the white blood cells (WBCs). After a washing with PBS, the cells were analyzed by flow cytometry using the FACSCanto II (BD Biosciences). The antibodies used in this study were as follows: PE-conjugated an-ti-CD16 mAb (3G8) and APC-conjugated anti-HLA-DR mAb (G46-6) from BD Biosciences; Brilliant violet 421-conjugated anti-CD33 mAb (WM53) from BioLegend; APC-conjugated anti latency-associated peptide-1 (LAP; the N-terminal region of transforming growth factor-β1 precursor) mAb (#27232) from R&D Systems (Minneapolis, MN, USA); and PE- or FITC-conjugated anti-GPI-80 mAb (3H9) from MBL (Nagoya, Japan). For isotype-matched control mAbs, IgG1 (MOPC-21) and IgG2a controls (G155-178) were obtained from BD Biosciences. The mean fluorescence intensity (MFI) and robust CV were analyzed using FlowJo software version 7.8.6 (TreeStar, Ashland, OR, USA) [10 (link)].