Immunophenotyping of Plasmodium falciparum

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).