Dengue Virus Infection and Extracellular Vesicle Isolation in Aedes aegypti Cells

Aedes aegypti (ATC-10) (ATCC^® CCL-125™, Manassas, VA, USA) cells were cultured in DMEM (Gibco™ 11995065) containing 10% FBS (Gemini 100–106), 1% penicillin/streptomycin (Gibco™ 15140163), and 1% tryptose phosphate broth (Gibco™ 18050039) at 30 °C with 5% CO₂. The cells were treated with Dengue Virus type 2, New Guinea Strain (DENV-2-NGC) at MOI of 1.0. Following 2 h of treatment with DENV-2, the media and virus were aspirated, and the cells were washed three times with sterile PBS (Gibco™ 10010049) and cultured in DMEM containing 10% exosome-depleted FBS (Gibco™ A2720803, Thermo Scientific, Waltham, MA, USA), 1% penicillin/streptomycin (Gibco™ 15140163), and 1% tryptose phosphate broth (Gibco™ 18050039, Thermo Scientific, Waltham, MA, USA) for 72 h. The cell supernatants following infection or not were collected and used to isolate extracellular vesicles using Invitrogen™ Total Exosome Isolation Reagent (Invitrogen™ 4478359, Thermo Scientific, Waltham, MA, USA), a method previously described for the isolation of EVs from Aedes cells [27 (link),55 (link)], according to the manufacturer’s instructions. The proteins contained in these EVs were processed using the Thermo Scientific Pierce In-Solution Tryptic Digestion and Guanidination Kit (Thermo Scientific™ 89895, Waltham, MA, USA) according to the manufacturer’s instructions.