HPASMCs were seeded at 5 × 103 cells per well in a 96-well plate and treated under different conditions. Numbers of cells were counted in MTT assay (Amresco, USA) as described before54 (link). For analysis of DNA synthesis, a sample of 1 × 106 cells were incubated with BrdU (10 μM) at 37 °C for 1 h. Collected cells were fixed and permeabilized, followed by exposure to DNase (300 μg per sample) at 37 °C for 1 h. The incorporated BrdU was stained with specific anti-BrdU fluorescent antibody at room temperature for 20 min. 7-AAD (20 μL per sample) was added for staining of total DNA. All reagents were from BD Pharmingen, USA. Detected cells in S phrase (P4), G0/G1 phrase (P3), G2 phrase (P5) and apoptosis phrase (P6) were separated and analyzed by an LSRFortessa flow cytometer (Becton Dickinson, USA). S phrase cells at a cell cycle was calculated as the percent of P4/(P3 + P4 + P5).
Mtt assay
Manufactured by Avantor
Sourced in United States, China
The MTT assay is a colorimetric method used to measure the metabolic activity of cells. It involves the conversion of the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) into a formazan product by the mitochondrial enzymes of viable cells.
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Lab products found in correlation
Softmax apparatus, by Molecular Devices (3 mentions)
Microplate reader, by Agilent Technologies (3 mentions)
96 well plate, by Corning (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Berberine, by Merck Group (1 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Enspire multimode plate reader, by PerkinElmer (1 mentions)
96 well plate, by SPL Life Sciences (1 mentions)
Xcelligence rtca dp, by Agilent Technologies (1 mentions)
Celltiter glo luminescent cell viability assay, by Promega (1 mentions)
Infinite m200, by Tecan (1 mentions)
Spectrophotometer microplate reader, by Bio-Rad (1 mentions)
Prestoblue assay, by Thermo Fisher Scientific (1 mentions)
1420 multilabel counter, by PerkinElmer (1 mentions)
Dimethyl sulfoxide (dmso), by Solarbio (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Tecan (1 mentions)
Alamarblue assay, by Thermo Fisher Scientific (1 mentions)
Celltiter glo assay, by Promega (1 mentions)
32 protocols using mtt assay
1
Quantifying Cell Proliferation Using BrdU Assay
2
Quantifying PDLC Proliferation via MTT Assay
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Proliferation of PDLCs following various treatments was determined by MTT assay (Amresco, LLC, Solon, OH, USA). A total of 320 µl MTT solution (5 g/l dissolved in ddH2O) was added to each well of a 6-well plate and cells were incubated at 37°C for 4 h. The MTT solution and culture medium was removed and 3,000 µl dimethyl sulfoxide was added. Complete dissolution of each sample was aided by 10 min of light-tight vibration. The absorbance was measured at a wavelength of 490 nm using a spectrophotometer. Pure culture medium without PDLCs was used as the blank control.
3
Berberine's Cytotoxic Effects: MTT Assay
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Cell viability was determined using MTT assay (Amresco, Solon, OH, USA). Briefly, 5×104/ml cells per well were plated in 96-well plates and incubated for 24 h. The cells were then treated with different concentrations (0, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 20 μM, 50 μM, 100 μM and 500 μM) of berberine (Sigma-Aldrich, St. Louis, MO, USA) for 24 h. Then the cells were treated with 20 μl of 5 mg/ml MTT and incubated for 4 h at 37°C. Then the medium was discarded, and 200 μl of dimethylsulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) was added. Absorbance was measured at 570 nm with an ELISA plate reader (Infinite® 200 PRO, TECAN, Männedorf, CH). The viability of berberine-treated cells was calculated by comparing to vehicle-treated cells, which were arbitrarily assigned 100%. The experiments were performed in triplicate, independently.
4
Evaluating oHSV2 Cytotoxicity in Cancer Cells
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The cytopathic effect was evaluated by viral cytotoxicity, cell cycle progression, and apoptosis analysis. The effect of oHSV2 on the proliferation of cancer cell lines was evaluated using the MTT assay (AMRESCO LLC, Solon, OH, USA) according to the manufacturer’s instructions. Cell viability was determined by measuring absorbance at 570 nm with a reference of 630 nm using a microplate reader (BIO-RAD, Hercules, CA, USA). The controls on each day were set at 100% viability. The percentage cell survival rate treated with oHSV2 was calculated using the following formula: (100%× [absorbance value of experimental cells]/[absorbance value of control cells]). All measurements were performed in triplicate.
5
Cell Proliferation Assay Using MTT
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Cell proliferation was analyzed using a MTT assay (Amresco, Shanghai, China). The cells were then seeded into 96-well plates (4 × 103 cells/well). At the appropriate time after transfection, 20 µL of MTT solution (5 mg/mL) was added to each well. After 3 h, media containing MTT solution were removed and 100 µL DMSO (Sigma-Aldrich, St. Louis, MO, USA) was added to each well. The absorbance at 540 nm was measured with a SoftMax apparatus (Molecular Devices, Sunnyvale, CA, USA). At least three independent experiments were performed to confirm the reproducibility of the results.
6
MTT Assay for Cell Viability
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Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Amresco Inc., OH, United States). The MTT assay was performed by addition of 0.5 mg/ml (final concentration) MTT to the cell media. Cells were incubated at 37°C until blue crystals were visible by eye (approximately 30–60 min). The media was removed and replaced with 100 μl of DMSO per well to dissolve the crystals. One hundred microliter aliquots were distributed to 96-well plate wells and the absorbance at 585 nm was determined using an EnSpire® Multimode Plate Reader (Perkin Elmer, Waltham, MA, United States). MTT reduction was blank corrected and expressed as a percentage of untreated controls.
7
MTT Assay for Cell Viability
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The anti-proliferative effects of EGCG and EC were investigated using a 3,-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (AMRESCO, Solon, OH, USA). In brief, cells were plated in a 96-well plate (SPL Life Sciences, Korea) at a density of 1 × 104 cells/well. After treatment, MTT solution (1 mg/ml in PBS) was added and incubated in a CO2 incubator at 37 °C for 4 h. The mitochondrial reductase enzyme of healthy cells caused purple formazan crystal formation. Then, DMSO was added to dissolve this crystal. Absorbance was detected with a microplate reader (BioTek Instruments, Winooski, VT, USA) (BioTek) at 595 nm wavelength. Percentages of cell viability were calculated and compared with the control by Graph pad prism version 5.
8
Adenoviral Infection and Cell Proliferation
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Cells were seeded in 24-well plates at 4 × 104 per well and infected with the indicated adenoviruses. Cell proliferation was assessed with MTT assay (Amresco, #0793). Specifically, cells were incubated with MTT at 37 °C for 4 h. The medium was then replaced with dimethyl sulfoxide and cells were solubilized. MTT cleavage was quantified using a spectrophotometer to measure absorption at 490 nm with absorption at 630 nm subtracted as background. CellTiter-Glo® Luminescent Cell Viability Assay (Promega, #G7570) was utilized to assess cell viability. For monitoring cell proliferation at real-time, 4 × 104 cells in a well of 24-well plate were subjected to the measurement by an impedance-based real-time instrument system (xCELLigence RTCA DP, ACEA Biosciences Inc.).
9
MTT Assay for Cardiac Fibroblast Viability
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The cardiac fibroblasts were seeded in 96-well culture plates. The MTT assay (Amresco, Solon, USA) was performed when the number of adherent cells reached 2 × 104 cells per well. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to test cell viability according to the manufacturer's protocols. The absorbance was calculated at 490 nm by Microplate Reader (Infinite M200, TECAN).
10
MTT Assay for Cell Viability
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Cell viability was determined by the MTT assay following the vendor’s protocol (Amresco, Cleveland, OH, USA). The SMMC-7721 cells were seeded at the initial cell density of 5 × 103 cells/well in 96-well plates and cultured with varying concentrations of DFP or VC for specified times. At harvest time, the MTT reagent (0.5 mg/mL) was added to all wells in the 96-well plate, followed by incubation for 4 hours. Following the incubation, the supernatant was removed from the wells and dimethyl sulfoxide was added to each well to dissolve the formazan crystals. The absorbance was then measured using a spectrophotometer microplate reader (Bio-RAD, Hercules, CA, USA) at a wavelength of 492 nm. The cell inhibitory rate was expressed as a percentage (%) of 1 minus the ratio of viable cell number after drug treatment relative to the control cell number. The IC50 (50% inhibition concentration, defined as the drug concentration resulting in 50% inhibition vs the untreated culture) of DFP and VC was calculated using the data generated from the MTT experiment. This experiment was repeated 3 times, and the results were statistically analyzed using the unpaired Student’s t test.
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