Fig. 3

Fluorescence intensity correlates with reprogramming factor transcription levels, and allows for discrimination between high and low levels of O, K, S, and M. a Gating strategy for discriminating high vs low fluorescence. Single cells out of 10,000 events are shown. Gates are drawn according to the process described in Methods. The top two FACS plots show single events. The bottom two plots are the ok and OK populations (low vs high OK expression), respectively (blue vs green plots). Gates delineating the sm and SM populations are shown. Percentages shown are relative to the parental gates. b Laser-scanning confocal images of transfected HFFs after sorting for negative, low fluorescence (oksm), and high fluorescence (OKSM) on day 6 confirm the output of FACS. c Quantification of exogenous mRNA by qRT-PCR. HFFs sorted for negative, low, and high fluorescence were assayed by qPCR directly after sorting. Each target was significantly different between groups, p < 0.005. Error bars represent SEM. R ² > 0.987 for all mRNA copy numbers vs mean fluorescence intensity values. d Mean fluorescence intensity (MFI) of individual sorts measured during flow vs mRNA copy number per cell measured by qRT-PR of corresponding Yamanaka/Fluorescent reporter sets. Each point represents one biological replicate (Color figure online). OKSM Oct3/4 + shp53, Klf, Sox2, and L-Myc + Lin28