Transwell Chamber Assay

Chemotactic migration of L929 cells was evaluated by transwell chamber assay as described previously (Yuan et al., 2019). Briefly, L929 cells (2 × 10⁴ cells in 200 μl culture media with 1% FBS) were seeded in the upper chambers (8 μm pore size; Corning, NY, United States). The lower compartments were filled with 500 μl culture media containing activin A (0, 5, 10, and 20 ng/mL) or CXCL12 (100 ng/mL). After incubation for 10 h, the cells on the upper side of chamber were removed with cotton-tipped swabs. Cells that had passed through the insert membranes were fixed with 4% paraformaldehyde for 20 min and stained with Giemsa. Experiments were repeated at least three times. The stained cells were imaged on an inverted microscope and cell numbers were counted in five randomly chosen fields from each chamber.