Culture Conditions and Recombinant Protein Expression in S2

Drosophila Schneider 2 (S2) derived from a primary culture of late stage Drosophila melanogaster embryos was purchased from Thermo Fisher Scientific Inc. (Catalogue no. R690-07). To increase the cell yield, the S2 cells were grown in Schneider’s Drosophila medium (Catalogue no. 11720-034) at 28 °C without CO₂ in suspension with spinners and shake flasks according to current protocols. Split cells at a 1:2 to 1:5 ratio were diluted into new culture every 3 to 4 days when the cells reached a density of 2 to 4 × 10⁶ cells/mL. This procedure maintained the S2 cells. The insect expression vector was stably cotransfected into S2 with pCoHygro using the calcium phosphate transfection method, and the transfectants were selected by 300 μg/mL hygromycin-B. Cell death was verified by Trypan blue staining. A 19:1 (w/w) ratio of expression vector to selection vector was used in co-transfection. Target recombinant protein was secreted into the medium and then collected by centrifugation for further purification.