Here we describe methodologies to characterize, delineate, and quantify pluripotent cells between naïve, formative, and primed pluripotent state mouse embryonic stem cell (mESCs) populations using flow cytometric analysis. This methodology can validate pluripotent states, sort individual cells of interest, and determine the efficiency of transitioning naïve mESCs to a primed-like state as mouse epiblast-like cells (mEpiLCs) and onto fully primed mouse epiblast stem cells (mEpiSCs). Quantification of the cell surface markers; SSEA1(CD15) and CD24 introduces an effective method of distinguishing individual cells from a population by their respective positioning in the pluripotent spectrum. Additionally, this protocol can be used to demarcate and sort cells via fluorescently activated cell sorting for downstream applications. Flow cytometric analysis within mESCs, mEpiLCs, and mEpiSCs can be efficiently completed using these optimized protocols.
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