Macs depletion column

The VarO and the 3D7/Pf13 parasite lines were described elsewhere^{21 (link),25 (link)}. The P. falciparum isolate NF54^{41 (link)} was used to generate clonal and transgenic lines (see Supplemental Table 1 for description of parasite lines). Parasites were cultivated in vitro as described^{42 (link)} using RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated human serum, hypoxanthin 10 mM, gentamicin (20 µg/ml) and human erythrocytes at 5% hematocrit. Infected erythrocytes were cultivated at 37 °C, 5% CO2, 2% O2. To obtain synchronous asexual stages, parasites were synchronized by the isolation of trophozoites by magnetic isolation using a MACS depletion column (Miltenyi Biotec) in conjunction with a magnetic separator, and placed back into culture. After the invasion of merozoites, ring-stage parasites were selected by magnetic depletion of trophozoites to obtain a tighter window of synchronization. Synchronous production of gametocytes was achieved as described^{43 (link)}. Briefly, the culture medium was supplemented with 50 mM N-acetyl-glucosamine from day 0 onwards, and a medium replacement was continued for 5 to 6 days to eliminate the asexual stages.

T cells and B cells were sorted from splenic cell suspensions from DO11.10 TCR transgenic mice on the BALB/c background with a typical purity of >90%. For CD4⁺ T cells, splenocytes were incubated with magnetic bead-conjugated antibodies against CD11b, CD11c, Ly6G, CD8, and B220 (Miltenyi Biotec, San Diego, CA), and the cells without magnetic beads were sorted using a MACS depletion-column (Miltenyi Biotec). Antibodies against CD11b, CD11c, Ly6G, CD8, and Thy1.2 were used for B cells. For T cell activation, CD4⁺ T cells were cultured on a plate pre-coated with antibodies against CD3 and CD28 (1 μg/ml each, from BD Biosciences, Franklin Lakes, NJ). All animal experiments were conducted according to the institutional guidelines for animal welfare as per protocols approved by the Animal Experiment Committee of Osaka University.

The P. falciparum clonal lines B10 and 3D7 (clones of NF54), and the transgenic lines pHL-pfpka-r, and PfPDEδ- clone 4 have been described elsewhere [25 (link),42 (link),53 (link)]. Parasites were cultivated in vitro under standard conditions using RPMI 1640 medium supplemented with 10% heat-inactivated human serum and human erythrocytes at a 5% haematocrit [54 (link)]. Synchronous production of highly specific gametocytes stages was achieved according to described protocol [55 (link)]. For the isolation of gametocytes, culture medium was supplemented with 50mM (final concentration) N-acetylglucosamine (GlcNAc) from day 0 onwards and medium replacement was continued for 5 days to eliminate the asexual stages. Gametocyte preparations were enriched in different experiments by magnetic isolation using a MACS depletion column (Miltenyi Biotec) in conjunction with a magnetic separator.

The P. falciparum NF54 strain, the B10 clone and the transgenic lines pHLpfpkar, PfPDEδ⁻, NF54-cg6-pfs16-CBG99 and NF54-pfs47-pfs16-GFP have been described elsewhere^{16 (link),23 (link),27 (link),42 (link)}. Parasites were cultivated in vitro under standard conditions using RPMI 1640 medium supplemented with 10% heat-inactivated human serum and human erythrocytes at a 5% hematocrit. To obtain synchronous asexual stages, parasites were synchronized by the isolation of schizonts by magnetic isolation using a MACS depletion column (Miltenyi Biotec) in conjunction with a magnetic separator, and placed back into culture. After invasion of merozoites, a second magnetic isolation was used for the selection of ring-stage parasites to obtain a tighter window of synchronization. Synchronous production of specific gametocytes stages was achieved by treating synchronized cultures at the ring stage (10–15% parasitemia, day 0) with 50 mM N-acetylglucosamine (NAG) for 5 days to eliminate asexual parasites. Gametocyte preparations were enriched in different experiments by magnetic isolation. Stage I GIE were collected at day 1 after initiating NAG treatment, stage II GIE were collected at days 2 and 3, stage III GIE were collected at days 4 and 5, stage IV GIE were collected at days 6 and 7, and stage V GIE were collected at day 8 onwards.