Male mice aged 8-15 weeks were infected by intravenous injection of viable P. chabaudi AS parasites (WT) or PccASluc (luciferase-expressing; [45 (link)]). To ensure viability of the parasites, a frozen aliquot was thawed and injected intraperitoneally into a transfer mouse. The number of asexual parasites intravenously injected into each mouse was adjusted according to body weight so that every animal received 1x104 iRBCs per 20 grams. Parasitemia was monitored from day 5 post infection every 48 hours by Giemsa-stained thin blood smear. Anti-GCSF antibody (150 μl per mouse, R&D) or isotype control (150 μl per mouse, R&D) were injected intravenously on day 7 post infection.
Mice were bled by cardiac puncture under non-recovery deep anesthesia. Blood was kept from coagulating by addition of 50 μM final concentration of EDTA (Sigma). Plasma was generated by centrifugation at 10,000 x g at 4°C for 10 minutes. Plasma was aliquoted, snap frozen in liquid nitrogen and stored at -80°C until further use. Plasma was always thawed on ice.
Organs were harvested without additional perfusion (except in parasite sequestration experiments) as blood was removed by terminal bleeding of the animals. The organs were fixed for 20h at room temperature in 2% PFA.
Mice were bled by cardiac puncture under non-recovery deep anesthesia. Blood was kept from coagulating by addition of 50 μM final concentration of EDTA (Sigma). Plasma was generated by centrifugation at 10,000 x g at 4°C for 10 minutes. Plasma was aliquoted, snap frozen in liquid nitrogen and stored at -80°C until further use. Plasma was always thawed on ice.
Organs were harvested without additional perfusion (except in parasite sequestration experiments) as blood was removed by terminal bleeding of the animals. The organs were fixed for 20h at room temperature in 2% PFA.