Igm fitc clone g20 127

Single-cell suspensions from the BM and peripheral blood were first incubated with human BD Fc block (BD Biosciences) to block Fcγ receptor and then stained with the following anti-human Abs: CD19-APC (clone HIB19, BioLegend), CD34-PE (clone 563, BD Biosciences), CD20-FITC (clone 2H7, BioLegend), CD27-APC/Cyanine7 (clone O323, BioLegend), IgD-PE (clone IA6-2, BD Biosciences), IgM-FITC (clone G20-127, BD Biosciences), CD43-PE (clone CD43-10G7, BioLegend), CD38-PE/Cy7 (clone HIT2, BioLegend), CD38-Percp/Cy5.5 (clone HIT2, BD Biosciences), CD24-PE (clone ML5, BD Biosciences), CD27-V450 (clone M-T271, BD Biosciences), IgD-BV510 (clone IA6-2, BD Biosciences), IgG-PE/Cy7 (clone G18-145, BD Biosciences), Ki-67–PE (clone Ki-67, BioLegend), CD80-FITC (clone L307.4, BD Biosciences), CD86-PE (clone IT2.2, BD Biosciences), HLADR-PE (clone G46-6, BD Biosciences) and CD69-PE/Cy7 (clone FN50, BD Biosciences). 7-AAD Viability Staining Solution (eBioscience, Thermo Fisher Scientific) was used for live versus dead cell discrimination. Samples were analyzed with a FACSVerse flow cytometer (BD Biosciences) using the FACSuite software. Data analysis was performed with FlowJo 10 software (Treestar).

Non-human primate (NHP) peripheral blood mononuclear cells (PBMCs) were stained with LIVE/DEAD Fixable Violet Stain (ThermoFisher, Waltham, MA) for 10 min at room temperature. Cells were washed with Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco) with 4% fetal bovine serum (FBS) and incubated with CD3-BV421 (clone SP34–2, BD Biosciences), CD4-BV421 (clone OKT4, BioLegend), CD8-BV421 (clone RPA-T8, BioLegend), CD14-BV421 (clone M5E2, BioLegend), CD20-Cy5.5PerCP (clone 2H7, BioLegend), IgG-A700 (clone G18–145, BD Biosciences), IgM-FITC (clone G20–127, BD Biosciences), FP9-PE, and FP9-APC-Cy7 for 20 min at room temperature. The two FP probes, FP9-PE and FP9-APC-Cy7, were made by conjugating biotinylated nine amino acid HIV-1 fusion peptide (AVGIGAVF) to fluorescence labeled streptavidin and used for staining FP-specific cells. Cells were then washed two times with RPMI with 4% FBS and assessed by flow cytometry. FP double-positive IgG+ memory B cells (CD3⁻CD4⁻CD8⁻CD14⁻CD20+IgG+IgM-FP9+) were sorted into 384-well culture plates at single-cell per well using a FACSAria II sorter (BD Biosciences), and relevant index information recorded. We carefully recorded all cell events during sorting and used the exact numbers of events from the sorter to calculate percentage of probe-stained cells in total IgG+ B cells.