2.5. Flow cytometry and cell sorting

The hiMSCs were trypsinized and resuspended in the fresh culture medium. The percentage of EGFP-positive cells was determined using a Gallios flow cytometer (Beckman Coulter, California, USA) at 488 nm excitation. Cell sorting was performed using a MoFlo XDP cell sorter (Beckman Coulter) at 488 nm excitation. EGFP purity and positivity in the sorted fractions were visually verified by using a fluorescence microscopy Nikon ECLIPSE Ti-U (Nikon CORPORATION, Tokyo, Japan) and evaluated by flow cytometric analysis (Gallios). For parental hiMSCs, forward versus side scatter gating was used for cell sorting as the control. The sorted cells were cultured in osteogenic differentiation medium containing VD3 for 3–4 days prior to purification of total RNA.