The cell viability was determined by using the MTT Cell Proliferation Assay (Beyotime, Shanghai, China). Briefly, cells were seeded in 96-well plates and incubated for 48 hours in DMEM, in the presence or absence of TMAO. Then 10 μL tetrazolium salt (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT) was added to the cells in phenol red-free culturemedium and incubated for 4 hours at 37°C. After dimethyl sulfoxide was added, the absorbance was measured at wavelength of 570 nm using a microplate reader (Thermo, Massachusetts, USA) within 1 hour.
Mtt cell proliferation assay
Manufactured by Beyotime
Sourced in China
The MTT cell proliferation assay is a colorimetric assay used to measure the metabolic activity of cells. It is based on the reduction of the yellow tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to purple formazan crystals by metabolically active cells. The amount of formazan produced is directly proportional to the number of viable cells, and can be quantified by measuring the absorbance at a specific wavelength.
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4 protocols using mtt cell proliferation assay
1
MTT Assay for Cell Viability
2
Assessing H2O2-Induced Cell Viability
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Cell viability was determined by MTT cell proliferation assay according to the manufacturer’s instructions (Beyotime, Jiangsu, China). Briefly, BmN-SWU1 cells were cultured in 96-well plates and treated with various H2O2 concentrations for 0, 8, 12, 16 or 24 h. Then, 10 μL of the MTT stock solution (5 mg/mL) was added to each well. After incubation for 4 h at 27°C, the MTT solution was removed and 100 μL formazan solution was added to each well. The cells were incubated for an additional 4 h at 27°C and the absorbance of the cell culture supernatant was measured at 570 nm using a microplate reader (Promega; Madison, WI, USA) to determine cell viability. Each assay was repeated three times.
Morphology of cells in culture was determined by fluorescence microscopy. First, H2O2 treated cells were fixed in 4% paraformaldehyde (PFA; Sangon, Shanghai, China) for 10 min at room temperature. After removal of the PFA solution the cells were washed with phosphate-buffered saline (PBS) three times for 5 min each. Then, PBS was removed and the cell nuclei were stained with DAPI (Beyotime) in the dark for 8 min. Finally, cell morphology was examined through a fluorescence microscope (Nikon, Tokyo, Japan) at an excitation wavelength of 360 nm.
Morphology of cells in culture was determined by fluorescence microscopy. First, H2O2 treated cells were fixed in 4% paraformaldehyde (PFA; Sangon, Shanghai, China) for 10 min at room temperature. After removal of the PFA solution the cells were washed with phosphate-buffered saline (PBS) three times for 5 min each. Then, PBS was removed and the cell nuclei were stained with DAPI (Beyotime) in the dark for 8 min. Finally, cell morphology was examined through a fluorescence microscope (Nikon, Tokyo, Japan) at an excitation wavelength of 360 nm.
3
Cytotoxicity Evaluation of SPACE-EGF
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The cytotoxicity of SPACE-EGF was determined using an MTT cell proliferation assay (Beyotime, Beijing, China). Cells were incubated with 1 mg/mL of SPACE-EGF in media for 6, 12, or 24 h. Untreated cells with liposome (Invitrogen, USA) were used as the control group. Viability was determined using a microplate reader (ELx800; Biotech Instruments Inc., USA) according to the manufacturer’s instructions.
4
Adenovirus-mediated HES1 Downregulation in NHEK Cells
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Adenovirus (AD) vector-mediated downregulation of human HES1 and scrambled control were constructed, amplified, and purified by Hanbio Biotechnology (Shanghai, China). NHEK cells were infected with the adenovirus at a multiplicity of infection of 100. After 2 days, NHEK cells were subjected to western blot, or MTT cell proliferation assay (Beyotime cat. C0009S). About 1.5 × 104 NHEK cells were seeded in the 96-well plate. The next day, 10 µl MTT solutions from the Stock (5 mg/ml) was added and cells were incubated in CO2 incubator in the dark for 4 h. 100 µl Formazan dissolved solution was added and cells were incubated in CO2 incubator in the dark for 3 h. The absorbance was read at 570 nm on a Multiskan GO (Thermo Fisher Scientific).
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