Fig. 2.
Cell viability and silencing efficiency depend on transfection conditions. a Scheme of the optimal gene silencing experiment: plating of cells (4.5 × 105 cells/well) and start of transfection at 0 h. Transfection complexes remained in the growth medium for 24 h until the medium exchange that was repeated at 48 h. At 48 h and 72 h time points, OCT4 level was analyzed by flow cytometry. Symbols: red line—transfection, black triangle—plating cells and adding transfection complexes, gray triangle—flow cytometric analysis, white triangle—medium exchange. b Flow cytometric analysis of OCT4 expression at 72 h in untreated cells or cells treated with 30 nM siOCT4 or siCtrl presented as percentage of OCT4-positive cells. X-axis represents the number of the cells plated into a well of 6-well plate. c Cell count normalized to untreated sample at 72 h time point while using optimal protocol. Abbreviations: UT, untreated. Statistical significance with P values less than 0.05 are labeled as * (mean ± SEM, N = 3)