Nanofil 100 μl 33g beveled needle

PbA^EEF1a-GFP sporozoites were inoculated (i.d.) in the ear pinna (750 sporozoites in 20–30 μl; 1% BSA in PBS) or i.v. in the retro-orbital vein (150 sporozoites in 50 μl; 1% BSA in PBS) using a microsyringe (Nanofil 100 μl; 33G beveled needle; World Precision Instruments).

Mosquitoes were narcotized (−20°C; 3-5 min), washed in 70% ethanol (1X; 10-20 s) and PBS (3X; 10-20 s; Ambion). Salivary glands were obtained by dissection under a zoom stereomicroscope (3X magnification; Nikon SMZ800, Japan) and preserved in RPMI 1640 medium (GIBCO BRL) or PBS. Salivary glands were smashed using Pellet pestles cordless motor (10-15 s; Sigma) to release sporozoites and centrifuged (100 g; 5 min) in LoBind microfuge tubes (Eppendorf). Supernatant was filtered (100 μm cell strainers; BD Falco) to exclude debris. Sporozoites were counted using KOVA Glasstic Slide 10 with quantitative grid (Fisher Scientific GMBH). PbA^EEF1a-GFP sporozoites were inoculated i.d. in the ear pinna (750 sporozoites in 20-30 μl; 1% BSA in PBS) or i.v. (retro-orbital; 150 sporozoites in 50 μl; 1% BSA in PBS) using a microsyringe (Nanofil 100 μl; 33G beveled needle; World Precision Instruments).