Please note! This essay has been submitted by a student.
S2 cells are derived due to the spontaneous immortalization of tissue broken down from late-stage Oregon-R embryos (Schneider, 1972 ). Cells are acquired as frozen stock from Drosophila Genomics Resource Centre (DGRC) and revived as per instructions on their website. Schneider’s Insect medium (Sigma Cat. #S0146) enriched with 10% fetal bovine serum (FBS, Gibco Cat. #10270) and 1% Penicillin-Streptomycin (Invitrogen Cat. #15070-063) is used as culturing media. Transfection was conducted in the absence of additional sera. In both cases, cells were maintained at 27°C in an incubator without CO2. Cells are passaged for maintenance every four days at a fresh media to cell-suspension ratio of 5 to 1. Only cells having gone through three successful passages are used for transfection assays.
S2 cells at full confluency in T-75 flasks are resuspended and counted. 1 x 105 cells/ml and 2 x 104 cells/ml were seeded into 6-well and 24-well plates, respectively. Plates are returned to the growth incubator for 20 to 24 hours prior to transfection. For assays involving DON-treatment, 4ug/ml of the drug is added to wells after this seeding period, and incubated for an additional 16 hours. Media containing DON is completely removed before transfection assays could commence. Effectene® (Qiagen Cat. #301425) is used exclusively as the transfection reagent in ratios recommended within manufacturer’s instructions, downloadable from Qiagen’s website as a manual. Each instance of plasmid introduction is followed by an incubation period of 48 hours.
For assays requiring immunofluorescence as means for observation, wells are lined with circular untreated cover slips of a suitable size prior to seeding. Immunostaining of cells is conducted with minor modifications to the protocol as described by abcam©. All rinses are performed with ice-cold 0.2% Triton-X in PBS (PBT), instead of just PBS. Transfection media is removed completely prior to first rinsing. Cells are fixed with fresh, room-temperature 4% PFA for 10 minutes. After three-successive quick rinses, cells are permeabilized with an extended 20-minute incubation with PBT. Enough BSA is added to each well for a final w/v of 1%, and left for at least an hour. Primary antibodies are added and nutation occurs overnight at 4°C. Cells are rinsed thrice, for five minutes per time, before secondary antibodies suspended in 1% BSA-PBT is added. After standing for two hours at room temperature, cells are rinsed, and slips are mounted on a drop of SlowFade™ Diamond Antifade Mountant (Cat #S36963, InvitrogenTM, ThermoFisher Scientific, USA). The sources of all primary and secondary antibodies used on cells can be found in previously described tables. Concentrations are reduced to a fourth of those applied on whole tissues.