Bullet blender 24 mixer mill

To determine the ZIKV infection and dissemination rates in Ar. subalbatus females, 15–25 female mosquitoes were sampled at 4, 7, and 10 days post exposure (dpe). To prevent cross-contamination of virus between the midgut, salivary glands, and ovary of each mosquito, these organs were carefully dissected using fresh dissecting needles, and the organs were iteratively rinsed in phosphate-buffered saline (PBS) three times each. The midgut, salivary glands, and ovaries from each mosquito were individually transferred to 1.5-mL microcentrifuge tubes containing 100 μL of Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO^TM, Invitrogen, Beijing, China) supplemented with 2% fetal bovine serum (FBS). These organs were homogenized using 5-mm stainless steel grinding balls (Next Advance, Averill Park, NY, USA) in a Bullet Blender^TM 24 mixer mill (Next Advance) set at a frequency of 12/sec for 1 min. All dissecting needles were dipped in 80% ethanol, burned, and cleaned before re-use.

To determine the ZIKV infection and dissemination rates in Ae. aegypti, 10–15 female mosquitoes were sampled at 2, 4, 6, 8, 10, 12, 16, 20 and 24 days post exposure (dpe). To prevent cross-contamination of virus between the midguts, salivary glands and ovaries of each mosquito, these organs were carefully dissected using fresh dissecting needles, and the organs were iteratively rinsed in phosphate-buffered saline three times each. The midguts, salivary glands and ovaries from each mosquito were individually transferred to 1.5 mL microtubes containing 100 μL of Dulbecco's modified Eagle's medium (DMEM; GIBCO, Invitrogen, Beijing, China) supplemented with 2% fetal bovine serum (FBS). These organs were homogenized using 5 mm stainless steel grinding balls (Next Advance, Averill Park, NY, USA) in a Bullet BlenderTM 24 mixer mill (Next Advance) set at frequency of 12/s for 1 min. All dissecting needles were dipped in 80% ethanol, burned and cleaned before reuse.