8 mm pore size insert

Fetal bovine serum (10% FBS) was used as a chemoattractant and was placed in the lower chamber of 24-well plates, while unprimed or cytokine-primed hUC-MSCs were plated at 5 × 10⁴ cells/well in the upper compartment of 24-well transwell inserts (8-mm pore size insert; Millipore, Billerica, MA, USA), and the plates were then incubated for 24 h at 37 °C under 5% CO₂. After the incubation, the transwell inserts were discarded, and the upper side of the filter was gently swabbed to remove the nonmigratory cells. Migrated cells on the lower side of the insert filter were then quickly fixed using 4% paraformaldehyde (PFA) and stained with 0.5% crystal violet for 20 min. Microscopic examination was performed, and five low-power fields (magnification, × 40) were randomly selected from each chamber. All the experiments in each group were performed in triplicate. The migrated cells were counted by two individuals in a blinded fashion.

Cultured Nestin⁺ and Nestin⁻ BMSCs were seeded on the lower chamber of the 24-well plates, while Sca-1⁺ CECs were plated at 5 × 10⁴ cells/well on the upper compartment of 24-well transwell inserts (8-mm pore size insert, Millipore, Billerica, MA, USA), and the plates were then incubated for 12 h at 37 °C under 5% CO₂. In the control group, no cell was seeded on the lower chamber. After the incubation, the transwell inserts were discarded, and the upper side of the filter was gently swabbed to remove the non-migratory cells. Migrated cells on the lower side of the insert filter were then quickly fixed using 4% paraformaldehyde (PFA) and stained with 0.5% crystal violet for 20 min. Neutralization assays were performed in a transwell system supplemented with anti-TIMP-1 (1.5 μg/ml; R&D Systems), anti-TIMP-2 (3 μg/ml; R&D Systems), and anti-CXCL12 (100 μg/ml; R&D Systems). Microscopic examination was performed and five low-power fields (magnification, × 400) were randomly selected from each chamber. All the experiments in each group were performed in triplicate. The migrated cells were counted by two individuals in a blinded fashion.