Jc 1 fluorescent probe assay kit

Ultrastructural analysis of mitochondria was performed using transmission electron microscopy (TEM). Cell pellets were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 2 h. Samples were placed on 100 mesh copper grids, post-stained with 1% uranyl acetate and 0.4% lead citrate, and examined at 80 kV using a Tecnai G2 Spirit Twin Transmission Electron Microscope (FEI Company). Mitochondrial membrane potential was measured using a JC-1 fluorescent probe assay kit (Beyotime Biotech). JC-1 work reagents were prepared according to the manufacturer’s protocols. Cells were incubated for 20 min at 37 °C in the dark after adding reagents to the cell medium. Cell fluorescence was observed using an LSM 780 confocal microscope (Carl Zeiss).

hBMSCs in each group (P3, P8, P8-SHED-CM, P8-HGF 100, P8-SCF 10 and P8-H+S) were seeded into 6-well culture plates (5 × 10⁴ cells per well) and cultured for 3 days. Mitochondrial membrane potential was detected by a JC-1 fluorescent probe assay kit (Beyotime Biotech, China) according to the manufacturer’s protocol. Cell fluorescence was observed under fluorescence microscope (Cael Zeiss, Oberkochen, Germany). To obtain quantitative results, the fluorescence intensity was detected by flow cytometry (CytoFLEX, Beckman Coulter). Data were analyzed with CytExpert Software (Beckman Coulter).