At 80–90% confluence, the H9 hESCs from all the experimental groups were harvested using 1 mg/mL of collagenase IV and passed through 0.2 µm filter to ensure single-cell dissociation. After washing the hESCs with PBS, the cells were incubated with a blocking buffer containing 1% BSA and 2% FBS in PBS for 1 h on ice. Subsequently, cells were incubated with Alexa fluor 647-conjugated mouse monoclonal SSEA4 antibody and Alexa fluor 488-conjugated mouse monoclonal TRA-1-60 antibody for 1 h on ice. Finally, incubated cells were washed three times with blocking buffer; then, cells were evaluated for corresponding marker expression using flow cytometry.

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