**4.1 Materials and methods**

### **4.1.1 Mice**

BALB/c, NOD/SCID and NSG mice are purchased from Charles River. Immunodeficient mice were kept in sterile isolators. They are housed in sterilized cages equipped with filter tops during the experimentation, and are provided with autoclaved tap water and a γirradiated pelleted diet ad libitum. They are manipulated under pathogen free conditions using laminar flux hoods.

### **4.1.2 Human red blood cells**

Human whole blood is provided by the blood bank. Blood donors had no history of malaria and all the blood groups are used without observing any difference on parasite survival. Whole blood is washed three times by centrifugation at 900 ×g, 5 minutes at room temperature and buffy coat was separated in order to eliminate white blood cells and platelets. Packed huRBCs are suspended in SAGM (Adenine, Glucose and mannitol solution) and kept at 4°C for a maximum of 2 weeks. Before use huRBCs are washed three times in RPMI-1640 medium (Gibco/BRL, Grand Island, N.Y.) supplemented with 1 mg hypoxanthine per liter (Sigma, St Louis, MO) and warmed 10 minutes at 37°C.

#### **4.1.3 Parasites**

*P. falciparum* lines 3D7, UPA, and K1 are employed in the study, along with clinical isolates taken from a patient at Bichat Hospital, Paris (which was used the day after being sampled). The Uganda Palo Alto (UPA) strain employed is the Palo Alto Marburg line, used for all the experiments conducted. Parasite cultures are not synchronized and therefore a mix of various developmental stages was injected to infect mice. Parasites are maintained under *in vitro* conditions at 5% hematocrit at 37oC in a candle jar in complete culture medium (RPMI-1640 medium (Gibco/BRL), 35mM HEPES (Sigma), 24mM NaHCO3, 10% albumax (Gibco/BRL) and 1mg of hypoxanthine (Sigma) per liter. Parasite samples are cryopreserved using the glycerol/sorbitol method (Rowe et al., 1968). The cultures are controlled for mycoplasma contamination by using polymerase chain reaction (PCR) technique. A non-lethal rodent parasite strain *Plasmodium yoelii* XNL1.1 is preserved in 500μl aliquot of cryo-preserving buffer at -80°C at 22% parasitaemia. The strain is thawed at room temperature, diluted twice in RPMI-1640 medium followed by the injection of 50 × 106 parasite directly into the mice.
