Boyden chambers of 24 well plates

Approximately 5 × 10⁴ hBMSCs were seeded with DMEM/F12 medium on the upper Boyden chambers of 24-well plates (8 μm; Corning, Inc.) and approximately 5 × 10⁴ vascular cells (hUASMCs or hUVECs) were seeded with the culture medium with the different concentrations of TGFβ3 and 2% FBS on the lower chambers. After incubating plates for 6 hours at 37 °C, hBMSC migration was assessed using the crystal violet dye method.

Approximately 5 × 10⁴ hBMSCs were seeded with DMEM/F12 medium on the upper Boyden chambers of 24-well plates (8 μm; Corning, Inc., USA) and the culture medium with different concentrations of TGFβ3 and 2% FBS was placed into the lower chambers. After incubating plates for 6 hours at 37 °C, the cells of the upper chamber were fixed, stained with 0.5% crystal violet dye, and removed with a cotton swab. The cells migrating to the lower surface were photographed and counted under a microscope. For TβRI/II signaling pathway inhibition, hBMSCs were pretreated with SB431542 (15 μM; Selleckchem Inc., TX, USA) for 1 hour prior to the growth factor administration.