Cd20 fitc clone 2h7

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).

Normal GC-cells from tonsil (one sample) were collected at the Ear-Nose-Throat clinic at the University Hospital in Umeå, Sweden. A single-cell suspension of the tonsil was prepared as previously described [31 (link)]. Before sorting, the cells were thawed and washed in sterile PBS supplemented with 2% FBS, then stained with Zombie aqua (1:1000) (BioLegend, San Diego, CA), anti-human IgD-BV421 (clone IA6-2, BD Biosciences, Franklin Lakes, NJ), CD20-FITC (clone 2H7, BioLegend), CD38-PE (clone HIT2, BioLegend), and CD19-PE-CF594 (clone HIB19, BD Biosciences) for 30 min at RT. Cell sorting was performed on a BD FACSMelody (BD Biosciences) and viable GC B-cells were defined as CD19 + CD20 + CD38 + IgD-, with the sort rate < 1500 events/second. A total of 864,000 cells were sorted into RPMI-1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA), supplemented with 1% penicillin–streptomycin, 20 mM HEPES, and 50% FBS.