Calcium Phosphate Transfection of HEK 293Ts and Lipofectamine 3000 Transfection of S2 Cells

HEK 293Ts were transfected using the following calcium phosphate method: Per 1 mL of media of the cell culture to be transfected, 50 μL of 2x HeBS^{28 (link),29} buffer, 1 μg of each DNA construct, and H₂O up to 94 μL was mixed. 6 μL of 2.5mM CaCl₂ was added after mixing of initial components, incubated for 1:45 minutes at room temperature, and added directly to cell culture. S2 cells were transfected with Lipofectamine 3000 reagent (ThermoFisher) following the manufacturer’s protocol. Transfection mixture contained 10ng/μL of DNA, 1.5% Lipofectamine 3000 reagent, and 2% P3000 reagent, and was brought up to volume with Opti-MEM (ThermoFisher). Transfection mix was incubated for 15 min at room temperature and was then added directly to the S2 cells. 100 μL of transfection mix per 1 mL of cell culture media was used. The transfected cells were imaged 72 hr after the transfection (24 hr after promoter induction).