X tremegene sirna reagent

HeLa:EGFP64 cells^{12 (link)} were seeded at ~100 000 cells per well into 6-well plates (BD Falcon) and incubated overnight in DMEM growth media supplemented with 10% FBS and 2% penicillin/streptomycin at 37 °C in 5% CO₂. Transfections were performed with 50–200 nM of each SSO using 5 μL of X-tremeGENE siRNA reagent (Roche) in 1 mL of Opti-Mem (Invitrogen) at 37 °C for 4 h, at which point the transfection mixtures were removed from the cells and replaced with DMEM growth media. After trans-fection, UV irradiations were performed as described above followed by 24 h incubation. Cells were then trypsinized with 500 μL of TrypLE (Invitrogen), washed, and resuspended in 300 μL of PBS, pH 7.4, for fluorescent analysis. Flow cytometry was performed on a FACSCalibur (Becton-Dickinson) instrument (488 nm argon laser, 530/50 nm BPF) and analyzed using Cellquest Pro software. Cells were gated for EGFP fluorescence (above 10^2.5 RFUs) and analyzed until 20 000 cells had been counted for each condition tested. The frequency of EGFP positive cells (gated/total) was normalized to the noncaged control SSO. Error bars represent the standard deviation of experimental triplicates.

HeLa:EGFP654 cells were seeded at ~10 000 cells per well into black clear bottom 96-well plates (BD Falcon) and incubated overnight in DMEM growth media supplemented with 10% FBS and 2% penicillin/streptomycin at 37 °C in 5% CO₂. Transfections were performed with 50–200 nM of each SSO using 1 μL of X-tremeGENE siRNA reagent (Roche) in 200 μL of Opti-Mem (Invitrogen) at 37 °C for 4 h. After 4 h, the Opti-Mem transfection mixtures were removed from the cells and replaced with DMEM growth media.