Quantification of pluripotency transcription factor levels in embryonic stem cells by flow cytometry

Embryonic stem (ES) cell lines are derived from the inner cell mass of the pre-implantation blastocyst and are characterized by the ability to undergo indefinite self-renewal while retaining the potential to differentiate into each of the three primary germ layers. The ability of individual ES cells to self-renew or appropriately respond to differentiation signals is influenced by the intracellular level of a number of crucial transcription factors. It is therefore important to be able to reliably quantify the levels of these proteins in single cells. Here we present an intracellular staining technique for flow cytometry suitable for monitoring transcription factor expression in ES cells. We illustrate the application of this technique to the detection of Oct4 and Nanog proteins and the coupling of this approach with fluorescent reporters of gene activity.