Chemically defined lipid mixture 1

On day 17 post-mosquito feed, 12 mm diameter coverslips were coated in the wells of a 24-well plate with 0.01% w/v collagen in PBS and incubated under UV light at room temperature for 1 h. The collagen was removed, and coverslips were washed once with PBS. HC-04 (50,000 per well) were plated in 24-well plates on the collagen-coated coverslips in 500 μL media. Media used were “culture media” (CM): equal volumes MEM and F12 supplemented with 10% HIFBS, 15 mM HEPES, 20 mM sodium bicarbonate, 15 μM phenol red, 200 units/mL penicillin, and 200 μg/mL streptomycin (Sattabongkot et al., 2006 (link); Cui et al., 2009 (link)); and “DMEM-NoGlc:” DMEM without Glc (Life Technologies), supplemented with 1 mM sodium pyruvate (Life Technologies), 1% FBS (Corning), 200 units/mL penicillin, and 200 μg/mL streptomycin. Additional supplementations of the DMEM-NoGlc that were tested were 1 × MEM amino acids without L-glutamine (Sigma-Aldrich) and chemically defined lipid mixture 1 (Sigma-Aldrich, St. Louis, MO, United States; containing 4 ng/mL arachidonic acid; 20 ng/mL linoleic, linolenic, myristic, oleic, palmitic, and stearic acids; 0.44 μg/mL cholesterol, 4.4 μg/mL Tween-80, 140 ng/mL tocopherol acetate, and 200 μg/mL pluronic F-68).