Celltiter 96 non radioactive cell proliferation assay kit

Cell viability was assessed using a CellTiter 96 Non-Radioactive Cell Proliferation Assay kit (Promega, USA) according to the manufacturer's instructions. LN229 and H4 cells were plated in 96-well plates (2 × 10³ cells/well). After 24 hours, cells were transfected with shRNA plasmids and then cultured for 1 and 2 day. The medium in each well was replaced with 120 μl of complete medium containing 20 μl of MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tertrazolium, inner salt) solution, and the plates were incubated for 1 hour at 37°C. The absorbance (490 nm) was measured at the indicated time points. For colony formation assay, 24 hours post-transfection, cells were seeded into 12-well plates (10² cells/well) and cultured for 8–14 days. The cells were stained with 0.1% crystal violet at the end of time course and the number of colonies was counted.

The in vitro chemosensitivity was measured by dimethyl thiazolyl-2,5-diphenyltetrazolium bromide (MTT) assay (CellTiter 96^® Non-Radioactive Cell Proliferation Assay kit, Promega, Madison, WI, USA). Briefly, Cancer cells were inoculated into 96-well plates in 100 μL medium (10⁴ cells) per well and allowed to attach and grow for 24 h. To determine the anti-proliferative effect of GypLXXV, Rg₃(S), Rb₁ and GypXVII, various concentrations of ginsenosides diluted with the FBS-free medium were added into the wells. Then the cells were exposed to drugs for 48 h at 37 °C, after which, MTT was added to each well and cultured for an additional 4 h. The formed formazan was dissolved in 100 μL of solubilization/stop solution after aspirating the culture medium. The plates were shaken mechanically for 5 min and incubated for 1 h. After shearing for each well the optical density was immediately read on a microplate reader at a wavelength of 595 nm. Results are expressed as LC₅₀ which was analyzed by GraphPad Prism 5 program.

MTT assay was described previously [17 (link), 42 (link)]. Cells were treated with NCTD, vehicle, E2 or TAM. The proliferation was determined using a CellTiter 96 nonradioactive cell proliferation assay kit (Promega, Madison, MI, USA) according to the manufacturer’s instructions.

Cell proliferation rates were determined for each cell line using an MTT assay kit (Cell Titer 96 Non-Radioactive Cell Proliferation Assay kit, Promega) following the manufacturer's instructions. Briefly, 1 × 10⁴ cells were seeded into the wells of 24-well plates and grown at 37 °C for 0, 24, 48 or 72 h. At each time point, the cells were incubated at 37 °C for 4 h in a 150 μl MTT reagent, followed by 1 h in 1 ml of stop solution. The absorbance of each well was read by a spectrophotometer at 570 nm. Alternatively, cells were counted manually at twelve hour intervals using a hemacytometer. Dead cells were excluded using trypan blue.

For the viability assay, CT-26 (colon cancer) and MDA-MB231 (breast cancer) cells were plated in triplicate on 96 wells plate (5.10³/well). After treatment with indicated drugs, cells were incubated for 48 hours at 37°C and incubated with solution of 5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tetrazolium substrate (Sigma) for 30 minutes at 37°C prior analysis on a microplate reader at 570. The LC50 value was defined as the drug concentration causing 50% cell death (LD = lethal dose) compared to growth of the untreated control cells. For cell proliferation, cells were plated in triplicate on 96 wells plate (5.10³/well) under serum free conditions for 24 h. The starved cells were then incubated for 24h with fresh media containing the two selected compounds B1f and B3 that inhibit both ChT-L and PA activities of iCP and cCP. These two compounds were chosen based on reported studies describing that the simultaneous inhibition of ChT-L and PA activities by two selective inhibitors of these activities seemed to enhance the anticancer efficiency [55 (link)]. Proliferation level in cells was evaluated, as reported previously [56 (link)] using the Cell Titer96 non-radioactive cell proliferation assay kit (Promega) according to manufacturer’s protocol