Transwell cell culture chamber apparatus with 8 μm pore membrane

Migration ability of FLSs was measured in a Transwell cell culture chamber apparatus with 8 μm pore membrane (Costar, New York, NY, USA). Briefly, FLSs were seed at a density of 5 × 10⁴ mL⁻¹ in six-well plates, and were treated with Cyclopamine (10 μM), Purmorphamine (1 μM), or co-treated with U0126-EtOH and Purmorphamine cultured for another 48 h, respectively. Then, FLSs were trypsinized, collected, and re-suspended with serum-free medium. The cell suspension (5 × 10³ mL⁻¹) was loaded into the upper chamber of the Transwell insert. Medium containing 10% FBS (600 μL) was added to the lower compartment as a chemoattractant. After 8 h of incubation, the filters were removed and cells remaining on the upper surface of the membrane were removed with a cotton swab. The cells adhering beneath the membrane were fixed in 4% paraformaldehyde and stained with crystal violet for 30 min. Migration ability of FLSs was quantified by cell counts of five random fields at 100 magnifications in each membrane.

Migration ability of FLSs was measured in a Transwell cell culture chamber apparatus with 8 μm pore membrane (Costar, New York, NY, USA). Briefly, FLSs were seed at a density of 5 × 10⁴ cells/ml in six-well plates. Twelve hours later, FLSs were trypsinized, collected, and re-suspended with serum-free medium. The cell suspension (5 × 10³ cells/ml) was loaded into the upper chamber of the Transwell insert. Medium containing 10% FBS (600 μl) was added to the lower compartment as a chemoattractant. After 8 h of incubation, the filters were removed and cells remaining on the upper surface of the membrane were removed with a cotton swab. The cells adhering beneath the membrane were fixed in 4% paraformaldehyde and stained with crystal violet for 30 min. Migration ability of FLSs was quantified by cell counts of five random fields at 100 magnifications in each membrane.