Mtt cell proliferation and cytotoxicity assay kit

[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) determination was used to assess cell proliferation. The transfected cells were seeded in a 96-well plate, then 20 µL of 5 mg/mL MTT solution (MTT Cell Proliferation and Cytotoxicity Assay Kit, BOSTER, Wuhan, China) was added and incubated for 4 h. Finally, 100 µL of dimethyl sulfoxide was added. A microplate reader was then used to measure the optical density at 490 nm.
For the colony formation assays, transfected cells were divided into 6-well plates (1000 cells/well). After 2 weeks in culture, the cells were fixed with 75% ethanol and stained with 0.2% crystal violet. The colony formation rate was determined by counting the number of stained colonies.
The EdU immunofluorescence assay was performed using a Cell-Light EdU DNA Cell Proliferation Kit (RiboBio) according to the manufacturer’s instructions. Treated ESCC cells were incubated with EdU for 3 h. After fixation and permeabilization, anti-EdU reagents and DAPI were used for cell staining. Fluorescence microscopy was used to observe and obtain images.

Viability of mammalian Vero and insect KC cells was measured using the MTT Cell Proliferation and Cytotoxicity Assay Kit (Boster Biological Technology, Pleasanton, CA 9, USA) following the manufacturer’s instructions. Briefly, confluent cells (96-well plate format, 5 × 10⁴ cells/well, triplicates) were treated with 100 μL of medium containing the indicated concentrations of ATA (two-fold dilutions, starting concentration of 1000 μM). Plates were incubated at 37 °C or 29 °C for 24 h. Samples were treated with 15 μL of Dye Solution and incubated at 37 °C in a 5% CO₂ atmosphere for 5 h. Next, the cells were treated with 100 μL of Formazan solubilisation solution for 16 h, and absorbance at 570 nm was read using a microplate reader. The viability of compound-treated cells was calculated as a percentage relative to values obtained with mock-treated cells. Non-linear regression curves and the median cytotoxic concentration (CC₅₀) were calculated using GraphPad Prism software v. 8.0. (GraphPad Software, San Diego, CA, USA).