6 well cell culture dishes

To evaluate the migration ability of each CM, hADSCs (1 × 10⁵ cells) were seeded into 6-well cell culture dishes (Corning Inc.). The cells were incubated for 24 h and then scratched. The migration area was measured at 0, 12, and 24 h after scratching. The relative migration area was calculated based on comparison to the initial scratch areas.

The MIN6 cells were a gift from Dr. Junichi Miyazaki (Osaka University Medical School, Osaka, Japan). They were grown and maintained as described previously28 (link)36 (link). Briefly, cells were plated on 6 well cell culture dishes (Corning) at a density of 0.8 × 10⁶ cells per well. The cell culture medium was composed of DMEM (Invitrogen) supplemented with 15% heat inactivated FBS (Hyclone), 100 U/ml penicillin, 100 μg/ml streptomycin (Invitrogen), 2 mmole/l glutamine (Invitrogen) and 50 μM beta mercaptoethanol (Invitrogen). Cells between passages 28 and 35 were used for the experiments. Cells were grown at 5% CO₂ and 37 °C. Culture medium was exchanged at 48 hours following cell seeding. At that time, 1.0 μmole/l CdCl₂ or vehicle (PBS) were added.

Cells from cell lines or freshly isolated cells (2 × 10⁵) were cultured in 6-well cell culture dishes (Corning, NY, USA) and RPMI-1640 supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Recombinant exogenous S100A4 was added as indicated. If applicable, S100A4 was preincubated with a twofold molar excess of anti-S100A4 for 30 min at 4°C. S1P function was altered by using the S1P₁/S1P₃ antagonist VPC23019 (Cayman, Ann Arbor, MI, USA), S1P₂ antagonist JTE013 (Selleck, Shanghai, China), transforming growth factor-β (TGF-β) was purchased from R&D Systems (Minneapolis, MN, USA).