Ssea3

AP staining was performed according to the manufacturer's (Sidansai, China) instructions. For the immunofluorescence staining, cells were fixed with 4% formaldehyde in DPBS for 15 min, permeabilized with 1% Triton X-100 in DPBS for 15 min, and blocked with 2% bovine serum albumin in DPBS for 1 h. Thereafter, cells were incubated with primary antibodies for 1 h, including those antibodies Oct4 (1∶200, Abcam), Sox2 (1∶200, Cell Signaling), Nanog (1∶200, Abcam), TRA-1-60 (Millipore, 1∶200), TRA-1-81 (Millipore, 1∶200), SSEA1 (1∶50, Developmental Studies Hybridoma Bank), SSEA3 (1∶50, Developmental Studies Hybridoma Bank), SSEA4 (1∶50, Developmental Studies Hybridoma Bank), 5-methyl cytidine (5-mC, 1∶200, Abcam), 5-hydroxymethyl cytidine (5-hmC, 1∶200, Active Motif), H3K27me3 (1∶250, Millipore). Primary antibodies were detected using secondary antibodies conjugated to Alexa Fluor 488 (1∶500, Molecular Probes) and Alexa Fluor 594 (1∶500, Molecular Probes).

Cells were dissociated by treatment with 5 mM EDTA/DPBS for 3 min, followed by further treatment with a 0.05% trypsin/EDTA solution for 1 min. After two washes with DMEM/10% foetal bovine serum and one wash with staining buffer (0.1% bovine serum albumin/DPBS), 1 × 10⁵ cells were incubated for 30 min at 4 °C with primary antibodies diluted in staining buffer. The cells were then rinsed twice with staining buffer and incubated for 30 min at 4 °C with secondary antibodies diluted in staining buffer. Then, the cells were rinsed twice with staining buffer and counterstained with propidium iodide just prior to analysis. Fluorescence intensities were analysed on a FACSCanto flow cytometer (Becton Dickinson), and the FL-1 positive ratios in the propidium iodide-negative region were monitored using FACSDiva software. Primary antibodies against the following molecules were used: stage-specific embryonic antigen (SSEA)-3 [2 μg/ml; Developmental Studies Hybridoma Bank (DSHB)], SSEA-4 (1 μg/ml; DSHB), Tra-1-60 (1 μg/ml; Millipore), Tra-1-81 (1 μg/ml; Millipore), and SSEA-1 (2 μg/ml; DSHB). Anti-mouse Ig/FITC conjugated (10 μg/ml; Becton Dickinson) and anti-rat IgM/Alexa 488 conjugated (1 μg/ml; Molecular Probes) were used as secondary antibodies.