P. falciparum parasites were cultured at 37°C in RPMI medium
containing 0.5 % Albumax I (Life Technologies) in candle jars as described
previously [22 (link)]. Synchronization of
parasite blood stage forms was achieved by plasmagel flotation [23 (link)] followed by sorbitol lysis [24 (link)]. Growth inhibition assays were
conducted in triplicate in 100 µL culture volumes at 3% hematocrit, starting
with 1 % trophozoite stage parasites, supplementing the culture medium with 1:10
diluted sera from non-immunized mice or from proteoliposome-immunized mice.
Parasitemias were counted by flow cytometry using ethidium-bromide-stained
culture material after 24 h and 48 h growth, as described previously [25 (link)]. Growth inhibition was calculated by
comparing parasitemias of cultures treated with purified antibodies from mice
immunized against non-related proteins and those treated with antibodies from
mice immunized with proteoliposomes containing PfRH5-CD14-GPIrec. In
both cases, murine IgG antibodies from immunized mice were purified with protein
G-agarose resin (Sigma), according to the manufacturer’s instructions.
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