Quantifying Pluripotency Markers by FACS

Intracellular FACS quantification of Oct4 and Nanog protein levels was performed as previously described12 (link) with minor modifications. Briefly, cells were dissociated with 0.25% trypsin and fixed first in 0.25% PFA in ES medium then in 70% methanol in PBS for 1 h at 4 °C. Cells were permeabilized and blocked in PBS/BSA 1%/Triton X-100 0.1%/goat serum10% (GIBCO Life Technologies, cat. no 16210-064) for 30 min at RT. The staining was performed overnight at 4 °C in the same buffer containing the indicated antibodies: anti-Oct4 (Santa Cruz, cat. no sc-5279) and anti-Nanog (Bethyl, cat. no A300-397A) to a final concentration of 2.5 and 10 μg ml⁻¹, respectively. For the detection anti-mouse IgG-Alexa Fluor-647 (Invitrogen, cat. no A21236) and anti-rabbit IgG-Alexa Fluor-405 (Invitrogen, cat. no. A31556) were used for Oct4 and Nanog, respectively (1/200 for 2 h at RT).
For cell cycle analysis, trypsinized cells were washed with PBS, and cell nuclei DNA were stained with propidium iodide overnight using the DNA con 3 kit (Consul T.S.) and samples were analysed using the FACS Canto II (Becton&Dickinson).