Cd19 pe cf594 clone hib19

Immunophenotyping of P. falciparum was done according to the protocol described elsewhere [21 (link)]. Briefly, cryopreserved PBMCs were used together with Fc block and the following fluorochrome-conjugated monoclonal antibodies: CD19 PE-CF594-clone HIB19, CD20 V450-clone L27, CD27 PE-Cy7-clone M-T271 (all from BD), FcRL4 APC-clone 413D12 (Biolegend) and FITC-conjugated mouse anti-human IgG monoclonal antibody (BD Horizon). B cells specific for P. falciparum were identified utilizing carboxyl Quantum dots (Invitrogen) conjugated to extract of schizont- and trophozoite-stage parasites as described in detail by Lugaajju et al. [83 (link)]. The analysis was performed on a LSRII flow cytometer (Becton–Dickinson Immuno Cytometry Systems, San Jose, USA). To detect the proportion of B cells (defined as CD19+ cells) that were specific for P. falciparum, the cells were categorized as follows: IgG MBCs (CD19+CD20+CD27+FcRL4±IgG+), non-IgG+ MBCs (CD19+CD20+CD27+FcRL4±IgG−), naïve B cells (CD19+CD20+CD27−FcRL4±IgG−), plasma cells/blasts (CD19+CD20−CD27+FcRL4±IgG−), and atypical MBCs (CD19+CD20+CD27−FcRL4±IgG+). Data was processed using FLOWJO software (Tree Star Inc., San Carlos, and Ca, USA).

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.