Cd20 v450 clone l27

PBMC were phenotyped using a LSRII flow cytometer (Becton-Dickinson Immuno Cytometry Systems, San Jose, USA) as described [39 (link)]. The following fluorochrome-conjugated monoclonal antibodies were used for B cell phenotyping: CD19 PE-CF594-clone HIB19, CD20 V450-clone L27, CD27 PE-Cy7-clone M-T271, and IgG FITC-clone G18-145 (all from BD). B cells specific for P. falciparum were identified utilizing Quantum dots (Invitrogen) conjugated to extract of schizont- and trophozoite-stage parasites as described in detail previously [38 (link)]. Proportions of B lymphocytes (defined as CD19⁺ cells) specific for P. falciparum (Pf+) were defined in the following cell compartments: IgG positive memory B cells (IgG⁺ MBC) (CD19⁺CD20⁺CD27⁺IgG⁺), IgG negative memory B cells (non-IgG⁺ MBC) (CD19⁺CD20⁺CD27⁺IgG⁻), naïve B cells (CD19⁺CD20⁺CD27⁻IgG⁻), plasma cells/blasts (CD19⁺CD20⁻CD27⁺IgG⁻), and CD27⁻ MBC, including atypical memory B cells (CD19⁺CD20⁺CD27⁻IgG⁺). Data was processed using FLOWJO software (Tree Star Inc, San Carlos, CA, USA).

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).