Analysis of MSC proliferation was performed using BrdU labeling.24 MSCs were seeded onto 8-well chamber slides (Thermo Fisher Scientific, USA) at a concentration of 2 × 104 cells per well. After adherence, BrdU labeling reagent (Invitrogen, USA) was added to the medium at 1:100 for 48 h. The cells were then fixed with 70% ethanol, denatured with 2 N HCl, and stained with a BrdU Staining Kit (Invitrogen, USA) according to the manufacturer’s instructions. Fluoroshield mounting medium with DAPI (Abcam, UK) was used for counterstaining and mounting. The BrdU-positive cells in five fields of view from each sample were quantified using ImageJ software (National Institute of Health, USA) and are presented relative to the total number of cells.

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