*2.5. Ex Vivo Production of Ookinetes and Flow Cytometry Analysis*

Eight days before ookinete production, 200 µL of *P. berghei* CTRP-GFP (kindly provided by Dr. Inga Siden-Kiamos [15]) in cryopreservation solution (RBC pellet:Roswell Park Memorial Institute medium (RPMI, Gibco, Dublin, Ireland):30% glycerol in water, 1:1:2) was administered i.p. to a BALB/c mouse. Four days later, this mouse was the donor to infect i.p. with 5 × 10<sup>7</sup> parasitized red blood cells in 200 µL of PBS a second mouse that one hour before the infection had been pretreated i.p. with phenylhydrazine (120 µL of a 10 mg/mL solution in PBS). For ookinete production, up to 1 mL of blood carrying gametocytes was collected by intracardiac puncture and diluted in 30 mL of ookinete medium: 10.4 g/L of RPMI supplemented with 2% *w*/*v* NaHCO3, 0.05% *w*/*v* hypoxanthine, 0.02% *w*/*v* xanthurenic acid, 50 U/mL penicillin and 50 µg/mL streptomycin, 20% heat-inactivated fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA), 25 mM HEPES, pH 7.4. The culture was incubated for 24 h at 21 ◦C with orbital shaking at 50 rpm (modified from [16]).

To check heparin influence on fertilization, to 487.5 µL of culture in a well of 24-well plates was added 12.5 µL of PBS containing heparin at 20 or 0.2 mg/mL, to provide final heparin concentrations of 500 µg/mL and 5 µg/mL. Samples were taken at two different time points (just after extraction and after 1 h incubation), including a control consisting of PBS only, in three independent experimental replicates. Twenty-four hours later, samples were diluted 1:100 in PBS and analyzed in a LSRFortessaTM flow cytometer (BD Biosciences, San Jose, CA, USA) set up with the five lasers, 20 parameters standard configuration. The GFP positive ookinete population was selected and counted using 488 nm laser excitation and a 525/40 nm emission collection filter. BD FACSDiva software version 6.1.3 (BD Biosciences) was used in data collection, and Flowing Software 2.5.1 (Turku Centre for Biotechnology, Turku, Finland) was used for analysis.

For targeting assays, mature ookinetes were washed twice with PBS and incubated with heparin-Cy5 at 400 µg/mL in ookinete medium without FBS for 1 h. The sample was finally diluted 1:100 in the same medium containing 0.2 µg/mL Hoechst 33342 and events recorded with an Amnis® ImageStream®<sup>X</sup> Mk II cytometer (Luminex Corporation, Austin, TX, USA) using 375 nm, 488 nm, and 642 nm excitation lasers for Hoechst 33342, GFP and Cy5 signals respectively. Data were analyzed with IDEAS® 6.3 software (Luminex Corporation).
