Yellow pipet tip

Fourteen days post blood meal or 7 days post injection mosquitoes were anesthetized with 100% CO₂, and placed on a CO₂ pad. Mosquitoes that died within the 7 or 14 days incubation period were discarded. Mosquitoes were immobilized by removing their legs and wings with forceps. The proboscis of each mosquito was inserted into a 200 μl yellow pipet tip (Greiner Bio-One) containing 5 μl of a 1:1 solution of 50% glucose solution and FBS, for a minimum of 45 min. After salivation, the mosquito bodies were added to 1.5 ml Safe-Seal micro tubes (Sarstedt, Nümbrecht, Germany) containing 0.5 mm zirconium beads (Next Advance, Averill Park, NY, United States). Each saliva sample was added to a 1.5 ml micro tube (Sarstedt) containing 55 μl DMEM-supplemented with additional Fungizone (50 μg/ml; Invitrogen), and Gentamycin (50 μg/ml; Life technologies), hereafter named DMEM-complete. Mosquito bodies and saliva samples were stored at -80°C until further processing.

Fourteen days post blood meal, mosquitoes were anesthetized with 100% CO₂ and placed on a CO₂ pad. Mosquitoes that died within the 14 days incubation period were counted and discarded. Mosquitoes were immobilized by removing their legs and wings. The proboscis of each mosquito was inserted into a 200-μl yellow pipet tip (Greiner Bio-One, Kremsmünster, Austria) containing 5 μl of a 1:1 solution of 50% glucose solution and FBS, for 1 hour. After salivation, the mosquito bodies were added to 1.5 ml Safe-Seal micro tubes (Sarstedt, Nümbrecht, Germany) containing 0.5 mm zirconium beads (Next Advance, Averill Park, New York, USA). Each saliva sample was added to a 1.5-ml micro tube (Sarstedt) with 55 μl DMEM cell culture medium containing FBS, Penicillin and streptomycin supplemented with additional Fungizone (50 μg/ml; Invitrogen), and Gentamycin (50 μg/ml; Life Technologies, Bleiswijk, The Netherlands), hereafter referred to as DMEM-complete. Mosquito bodies and saliva samples were stored at −80°C until further processing.