Celltiter 96 non radioactive cell proliferation kit

To quantify cell proliferation, we seeded 5,000 HCT116 cells transduced with either control (pLKO.1) or USP54-specific shRNAs per well (n = 6) into 96-well plates. Next, a Cell Titer 96 Non-Radioactive Cell Proliferation kit (Promega Corp.) was used following the manufacturer's instructions.

Proliferation was measured by CellTiter96 nonradioactive cell proliferation kit (Promega Corp., Madison, WI) and MTT based assay, according to the manufacturer’s recommendations. Briefly, cells were seeded in a 96-well plate overnight and incubated with MDEs for 24 h. At the end of the treatment period, the medium was changed and incubated with the dye solution for 4 h, at 37 °C followed by solubilization in dimethyl sulfoxide and spectrophotometric measurement. The rate of formazan dye formation was determined by measuring the absorbance (570 nm–640 nm). The 570 nm–640 nm reading value is directly proportional to the number of living cells.

HSCs’ proliferation was measured using methylene blue assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay.
Methylene blue assay: For the methylene blue assay, cells were fixed in glutaraldehyde at a final concentration of 0.05% for 10 min at room temperature. After washing, the cells were stained with 1% methylene blue in 0.1 M borate buffer, pH 8.5, for 60 min at room temperature. The cells were then washed extensively and rigorously, to remove excess dye, and then dried. The dye taken up by cells was eluted in 0.1 M HCl for 60 min at 37 °C, and absorbance was monitored at 620 nm. The cells were photographed after fixation and methylene blue staining.
MTT assay: The MTT assay was carried out using the CellTiter96 nonradioactive cell proliferation kit (Promega Corp, Madison, WI, USA) and MTT-based assay, according to the manufacturer’s recommendations. After isolation, cells were seeded in a 96-well plate. At the end of the treatment period, the medium was changed, and cells were incubated with the dye solution for 4 h at 37 °C, followed by solubilization in dimethyl sulfoxide, and spectrophotometric measurement was undertaken. The rate of formazan dye formation was determined by measuring the absorbance (570 nm–640 nm). The 570 nm–640 nm reading value is directly proportional to the number of living cells.

To quantify cell proliferation, 5000 (in the case of HEK293T and HeLa), 2500 (in the case of U2OS, NCI-H661, and MDA-MB-231), or 2000 (in the case of HCT116) cells per well were seeded into 96-well plates, and incubated at 37 ^oC, 5% CO₂ for 5 or 10 consecutive days. Then, at the desired time points, a Cell Titer 96 Non-Radioactive cell proliferation kit was used following the manufacturer’s instructions (Promega).

The cells were seeded (1×10³ cells/well) in 96-well plates and then treated with phenformin the next day, at indicated concentrations, for 5 days [35 (link)]. After 5 days, cell viability was assessed using a CellTiter 96 Non-Radioactive Cell Proliferation kit (Promega; Madison, WI). The medium was replaced with 50 μl of 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 2.5 mg/mL) in SFM, followed by a 4 hour incubation at 37°C. The MTT solution was then replaced with 50 μl of DMSO, followed by incubation on a shaker to dissolve the formazan crystals. The absorption was measured with a microplate reader at 540 nm. The cell viability of each group, based on 8 parallel samples, was calculated relative to the controls, which were normalized to 100% survival.