Rpmi 1640 containing l glutamine

P. falciparum FCR3CSA, NF54CSA, and 7G8CSA strains expressing VAR2CSA were cultured under standard conditions in O+ RBC in RPMI 1640 containing L-glutamine (Invitrogen France) supplemented with 5% Albumax I, 5% Human plasma, 1× hypoxanthin, and 20 μg/mL gentamicin [52 (link)]. Parasitemia was routinely monitored by a thin blood smear fixed with 100% methanol for 1 min before staining in 10% vol/vol Giemsa (Sigma-Aldrich) in phosphate-buffered saline for 15 min. Genomic DNA extracted from parasite cultures was regularly tested for Mycoplasma contamination (look out Mycoplasm PCR detection kit; Sigma). Parasite cultures were routinely selected by gelatin flotation using Plasmion (Fresenius Kabi France) to maintain knob-positive parasites [53 (link)].

Purified naïve B cells were cultured in B-cell medium (RPMI 1640 containing L-glutamine; Invitrogen Life Technologies, CA, USA), supplemented with 10% fetal calf serum (FCS) (Invitrogen Life Technologies, Waltham, MA, USA), 10 mM 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid (HEPES) (pH 7.4; Sigma-Aldrich, St Louis, MO, USA), 0.1 mM nonessential amino acid solution (Sigma-Aldrich), 1 mM sodium pyruvate (Invitrogen Life Technologies), 60 mg ml⁻¹ penicillin, 100 mg ml⁻¹ streptomycin, 40 mg ml⁻¹ transferrin (Sigma-Aldrich), and 20 μg ml⁻¹ Normocin (InVivogen, San Diego, CA, USA); and stimulated with 100 ng ml⁻¹ CD40L alone (Enzo, Farmingdale, NY, USA) or with IL-4 (100 ng ml⁻¹), IL-21 (50 ng ml⁻¹; both Peprotech), or CpG 2006 (1 μg ml⁻¹, Invitrogen, Carlsbad, CA, USA), APRIL (500 ng ml⁻¹, Adipogen, San Diego, CA, USA), in the presence or absence of IL-4 and IL-21. For some experiments, B cells were labelled with division-tracking dye cell trace violet (CTV, Invitrogen).^{32 (link)} For phenotypic and functional analysis, cells were cultured in 96-well plates for 5 or 6 days, collected, stained with CD20, CD27, CD38, IgG, IgM, IgA and the proportion of isotype switched and differentiated antibody secreting cells determined as previously described.^{6 (link)} Secreted IgM, IgG and IgA levels were determined by Ig Heavy chain-specific immunoassays as previously described.^{6 (link)}

THP1-Dual cells (Invivogen, catalog (cat.) no. thpd-nfis) and THP1 cells were cultured in RPMI complete medium (RPMI1640 containing l-glutamine; Invitrogen, cat. no. 11875-085), 10% v/v FBS (Invitrogen, cat. no. 26400-044) and penicillin-streptomycin (Pen/Strep, Invitrogen, cat. no. 15070-063) at 37 °C, +5% CO₂. The MC38 cell line derived from C57BL/6 murine colon adenocarcinoma was acquired from the laboratories of James Hodge, PhD and Jeffery Schlom, PhD (National Cancer Institute, National Institutes of Health). The murine melanoma-derived B16-SIY cells (referred to as B16-SIY; engineered to express the model SIYRYYGL (SIY) antigen to enable immune monitoring) were acquired from the Gajewski lab (University of Chicago)^{47 (link)}. FreeStyle 293-F cells were acquired from Thermo Fisher (cat. no. R79007). Cell lines were regularly tested and maintained negative for mycoplasma.