Evaluation of KI efficiency by flow cytometry
72 hours post-transfection, cells were sub-cultured to 6 well plates at
a seeding concentration of 2×105 cells/ml. The
remaining cells were re-suspended in PBS supplemented with 10% FBS and
analyzed by FACSCalibur (Becton Dickinson, Franklin Lakes, NJ) to
quantify the EGFP+ and/or TagRFP657+
cells. Similarly, after another 3 days, cells were re-analyzed by flow
cytometry and the remaining cells were transfected for the sequential
strategy. Cells were gated by forward scatter versus side scatter plots,
and fluorescence threshold was set based on mock (no DNA control) to be
about 0.1 %.