Ab175429

A total of 1 × 10⁶ cells (WT ESCs, passage number 29) were required for optimal performance. The cells were fixed by adding 100 µl of 4% PFA to each sample for 15 min and then permeabilized by adding 100 µl of PBS and 0.3% Triton-X 100. The cells were stained with primary and secondary antibodies. The primary antibody was anti-Rex1 antibody (Abcam, ab175429) and the secondary antibodies were donkey anti-mouse IgG (H + L) Secondary Antibody (Life Technologies, A16018) and donkey anti-mouse IgG H + L (TRITC) preadsorbed (Abcam, ab7058).

EH-BES cells were separately cultured in N2B27 medium, serum medium, and 2i medium for 7 days. Then cells from each group were fixed and permeabilized using BD Cytofix/Cytoperm solution (BD Biosciences) according to manufacturer’s instruction. Flow cytometry assay was performed according to a standard protocol. Briefly, the cells were incubated with mouse anti-Rex1 (Abcam, ab175429) and goat anti-Oct4 (Abcam, ab27985) for 60 min at 4°C. Then the cells were incubated with Alexa Fluor 488 goat anti-mouse IgG (Invitrogen, A-11001) or rabbit-anti-goat IgG (Invitrogen, A-11078). Flow cytometry assay was carried out using FC500 flow cytometer (Beckman Coulter). Statistical analysis was performed by using SPSS 11.0 (IBM Corp. USA). The experiments were separately repeated for 3 times and results were presented as means and standard deviation (SD).