Migration Assay

Effects of designated cells on migration ability of endothelial cells were determined using a 24-well Millicell hanging cell culture insert (8 μm pore size membrane filters, Merck, United States). First, the lower chamber of the 24-well plate was filled with 600 μl cell suspension containing CD51⁺cMSC^con, CD51⁺cMSC^shSCF, and young CD51⁺ cells. Then, 100 μl endothelial cell suspension (5 × 10⁵ cells/ml) in serum-free medium was plated into the rehydrated upper chamber. The cells were incubated for 8 h under hypoxia condition (1% O₂). Thereafter, the inserts were fixed in 4% paraformaldehyde for 20 min and stained with crystal violet for 30 min or with DAPI for 5 min. Images of the cells on the lower surface of the filters were acquired by fluorescence and bright-field microscopy (BioTek, United States). The migrated cells were quantified in five randomly selected fields.