Mra 1000

For P. falciparum sporozoites, female A. gambiae (Keele strain) or A. stephensi (Liston strain) were fed through an artificial membrane on a blood culture containing either P. falciparum NF54 (BEI Resources, MRA-1000) or 3D7HT-GFP (BEI Resources, MRA-1029) gametocytes (mature gametocytes were adjusted to 0.3% in 50% human red blood cells (O+) containing 50% human serum (O+); please refer to the ethics statement) at the Malaria Research Institute at the Johns Hopkins Bloomberg School of Public Health as previously described^{58 (link)–60 (link)}. P. falciparum-infected mosquitoes were wing-clipped at Johns Hopkins University and transferred to Yale University for sporozoite cell traversal studies.

The Plasmodium falciparum parasite lines used in this study were all derived from the NF54 strain (originally isolated from an imported malaria case in the Netherlands in the 1980s) (BEI Resources catalog no. MRA-1000) (32 (link)). Asexual parasites were cultured in human blood (UK National Blood Transfusion Service) and RPMI 1640 medium containing 0.5% (wt/vol) AlbumaxII (Invitrogen) at 37°C as previously described (33 (link)). Asexual parasites were used to produce gametocytes by seeding asexual rings at 1% parasitemia and 4% hematocrit on day 0 and feeding the parasites once a day during 15 days (day 0 to day 14) in 3% O₂–5% CO₂–92% N₂ gas in RPMI 1640 medium complemented with 25 mM HEPES, 50 mg/liter hypoxanthine, 2 g/liter sodium bicarbonate, and 10% human serum (34 (link)).

P. falciparum strain NF54 was obtained through BEI Resources (MRA-1000 (Patient Line E), contributed by Megan G. Dowler). Briefly, NF54 strain parasites were maintained in an atmosphere of N₂/CO₂/O₂: 90/5/5 and complete RPMI medium containing RPMI 1640, 25 mM HEPES, 50 μg ml⁻¹ of hypoxanthine, and 0.3 mgml⁻¹ of glutamine (KD Biomedical, Columbia, MD) supplemented with 25 mM NaHCO3 (pH 7.3), 5 μgml⁻¹ of gentamicin, and 10% human serum (Interstate Blood Bank, Memphis, TN)^{68 (link)}. Sorbitol treatment (5%, 10–30 min at 37 °C) and/or MACS® LS columns (Miltenyi Biotec, Auburn, CA) were used for synchronization and parasite stages were quantitated using Giemsa-stained culture smears to determine parasitemia and total RBC.

Plasmodium falciparum was cultured in T25 Nunclon Delta closed cap culture flasks (Thermo Fisher, Waltham, MA) in RPMI 1640 supplemented with 25 mM HEPES, 0.1 mM hypoxanthine, 25 μg/ml gentamicin (all Thermo Fisher), 0.5% Albumax II (Gibco, Gaithersberg, MD), and 4.5 mg/ml glucose (MilliporeSigma, St. Louis, MO) at 37 °C in a 5% CO2, 5% O2 atmosphere at 5% hematocrit below 5% parasitemia. Red blood cells were obtained from anonymized healthy donors in a protocol approved by the National Institutes of Health Institutional Review Board.
NF54 obtained through BEI Resources (MRA-1000), NIAID, NIH as part of the Human Microbiome Project. The parasite lines EXP2-mNeonGreen^{14 (link)}, EXP2-mNeonGreen—PV-mRuby3^{15 (link)}, NF54attb³⁵ (originally obtained from the authors of ref. ³⁵ ) were described previously.