Purification of P. berghei Ookinetes

Ookinetes were produced using the P. berghei line PbRFP, a P. berghei ANKA line that constitutively expresses RFP. Two female Swiss mice were injected intraperitoneally with 200 µl phenylhydrazine (6 mg/ml in PBS) to stimulate reticulocytosis. Two days later, the mice were infected intraperitoneally with 20*10⁶ iRBC PbRFP. Mice were bled three days post infection and 500 µl blood was transferred to 10 ml ookinete medium (RPMI supplemented with 20% (v/v) FCS, 50 µg/ml hypoxanthine, and 100 µM xanthurenic acid, adjusted to pH 7.8 – 8.0) at 19 °C. After 22 h of culture, ookinete cultures were underlaid with 5 ml 55% Nycodenz/PBS and centrifuged for 25 min at 210 xg without brake. The interphase containing purified ookinetes was collected, washed in PBS and immediately plunge frozen for EM.