4.2. Transwell Migration Assay

ST2 cells (2.5 × 10⁴) or BM-MSCs (2 × 10⁴) were seeded on Millicell inserts (8 μm pore size; EMD Millipore, Billerica, MA, USA) coated with type I collagen (5 μg/mL; Nitta Gelatin NA Inc., Morrisville, NC, USA). After 6 h of incubation, the media was changed to DMEM (GE Healthcare Life Sciences) supplemented with 2% FBS and 100 U/mL penicillin. After overnight incubation, SP (300 nM), TGF-β (100 ng/mL for ST2 cells or 10 ng/mL for BM-MSCs), or SDF-1 (50 ng/mL) were added to the lower chamber of each well. After 12 h, the inserts were fixed using 4% paraformaldehyde in phosphate buffered saline (PBS) for 10 min at room temperature, washed, and then stained using 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, Carlsbad, CA, USA) for 10 min. Then the membrane inserts were mounted on slides using ProLong Gold antifade mounting solution (Invitrogen). Cells on either the lower or upper surface of the five randomly selected areas of each Millicell membrane were imaged using a Zeiss LSM 700 confocal microscope (Zeiss LSM700, Zeiss, Oberkochen, Germany) and experiments were done on duplicated samples for each experimental condition. The number of cells in each image was counted using Adobe Photoshop CS6 (Adobe Systems Incorporated, San Jose, CA, USA) and the number of migrated cells was determined as a percentage of the total cells on both sides of the insert. To evaluate the effects of pretreatment with SP, TGF-1, or SDF-1 on the migration of ST2 and BM-MSCs in response to either of the other two factors, the cells were serum starved for 18 h and treated with SP, TGF-β or SDF-1 for 12 h and then seeded as before for the migration assay. SP receptor antagonist, RP 67580 (10 nM) or CP-96345 (1 μM) was added to the ST2 cells or BM-MSCs prior to their SP pretreatment, respectively and then used in the migration assay as before.