Culturing and Transfecting Drosophila S2 Cells

Drosophila S2 cells were cultured in Schneider's Insect Medium (Gibco) supplemented with 10% FCS and antibiotics at 25°C (Schneider, 1972 (link)). pUAST vectors with actin5Ce-Gal4 drivers were cotransfected using Effectene (Qiagen) according to the manufacturer's instructions, and harvested 36-48 h after transfection. For immunofluorescence or time-lapse imaging, cells were replated on coverslips or glass-bottom dishes coated with Concanavalin A (Wako) and were allowed to spread for 1-2 h (Rogers et al., 2002 (link)). For drug treatments, cells were treated with 1 µM Latrunculin A (Wako) or 10 µM Colchicine (Wako) for 1 h before imaging. For immunofluorescence, cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS (PBS-T) for 15 min, and blocked with 5% skimmed milk in Tris-buffered saline (TBS). Primary and secondary antibodies were diluted in the blocking solution. After each antibody incubation, the coverslips were washed three times with PBS-T. The cells were mounted in Vectashield mounting medium (Vector Labs).