Mtt reagent

Cell viability was determined colorimetrically using the 3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) reagent (Duchefa, Haarlem, The Netherlands). The MTT assay procedures were performed as previously described [43 (link)]. Briefly, cells at the exponential phase were seeded (2 × 10⁴ cells/mL) in a 96-well plate. After treatment with SHH, 10 μL of 5 mg/mL MTT solution was added to each well and incubated for 2 h. The supernatants were then aspirated, the formazan crystals in each well were dissolved in 100 μL of DMSO for 10 min at room temperature, and the plate was read at 570 nm using a microplate reader (Molecular Devices, San Jose, CA, USA). Data are expressed as the optical density or percentage of viable cells compared to the control.
Real-time live cell imaging was performed using Cell Observer (Carl Zeiss, Jena, Germany). Briefly, the cells were seeded at the density of 1.5 × 10³ cells/well on Culture-Insert 2-well (ibidi GmbH, Martinsried, Germany) and incubated in a humidified atmosphere with 5% CO₂ at 37 °C for 48 h. The cells were treated with 100 µg/mL SHH, colivelin was pretreated for 2 h, and then transferred to the Cell Observer. The cells were maintained at 37 °C in 5% CO2. Images were captured at 10 min intervals for 48 h. Cells cultured in the medium without adding SHH were taken as a control.

Cell viability was examined by the MTT reagent according to the manufacturer’s protocol (Duchefa Biochemie, Haarlem, Netherlands). Log-phase cells were trypsinized into single-cell suspension, and cells (1 × 10³ cells) were seeded in 96-well plates. Cells were transfected after 24 h as described above. After 24 h, 90 μL of plain media and MTT were mixed and cultured for 2 h in a 37 °C humidified incubator. Then, 100 µL of dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) was mixed and OD was measured using an Asys UVM 340 microplate reader (Biochrom, Cambridge, UK).