E2-Orange and E2-Red/Green are noncytotoxic to HeLa cells under conditions of standard high-level expression. (A) HeLa cells were transiently transfected in 24-well plates for constitutive high-level expression of the indicated FP. Three wells per FP per day were analyzed by flow cytometry, in parallel with untransfected cells, and the average brightness of the viable fluorescent cells was measured. The highest signal for a given FP was normalized to 100 units. Error bars represent s.e.m. (B) Fluorescence intensity distributions for the same data were analyzed for 48 h (blue) and 120 h (red) post-transfection. Each data point is a binned value that represents the percentage of cells with fluorescence in a range centered about the data point.