Celltiter 96 aqueous one solution non radioactive mts cell proliferation assay reagent

The cell proliferation was measured using the CellTiter 96® Aqueous One Solution Non-Radioactive (MTS) Cell Proliferation Assay Reagent (Promega), according to the manufacturer’s instructions. The anchorage-independent growth via soft agar assay was assayed by protocol as described^{49 (link)}. For clonogenic/colony formation assay, 5000 cells per well were plated in 6-well plates and allowed to grow for about 7–10 days until colonies could be clearly seen. Plates containing colonies were gently washed with PBS and fixed with 3.7% formaldehyde for 10 min. Colonies were stained with 0.2% crystal violet solution in 10% methanol for 10 min, followed by repeated washing with PBS to remove excess stain. The colony formation was quantified using the ImageJ-ColonyArea plugin^{50 (link)}.

Cells were transiently transfected with conditions of interest in a six-well plate (day 0) and incubated overnight. On day 1, 1,500–2,000 cells/well were seeded in triplicate in a 96-well plate in 100 μl of culture medium. Once the cells adhered, cell proliferation was measured using the CellTiter 96 Aqueous One Solution Non-Radioactive (MTS) Cell Proliferation Assay Reagent (Promega). 20 μl of MTS reagent was added to each well and following incubation of 2 h the absorbance was measured at 490 nm using a plate reader. Cell proliferation was monitored for 5 d. For 3D proliferation assay, 10,000 cells/well were seeded in triplicate in an ultra-low-attachment 96-well plate and measured via CellTiter-Glo 3D Cell Viability Assay (Promega), according to the manufacturers’ instructions.