The in vitro antiproliferative activity of the target compounds and CA-4 was measured by a standard MTT (meilunbio®, China) assay. Cervical cancer (HeLa), gastric adenocarcinoma (SGC-7901) and breast cancer (MCF-7) were used, respectively. Cells were inoculated in 96-well plates at a density of 2 × 103/well. After 24 h, the target concentration of drug was added to each well and incubated for 72 h at 37 °C under 5% CO2. 20 μL of fresh medium containing 5 mg/ml MTT solution was added and incubation continued for 4 h. After removing the medium containing MTT from each well, 150 μL of dimethyl sulfoxide (DMSO) was added to each well until the purple formazan crystals was completely dissolved, placed in a multimode plate reader Victor Nivo 3S (Perkinelmer, USA) and the absorbance was measured at 490 nm.26 (link)
Methyl thiazolyl tetrazolium (mtt)
Manufactured by PerkinElmer
Sourced in United States
MTT is a colorimetric assay for assessing cell metabolic activity. It utilizes the tetrazolium dye MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to measure the reduction of metabolically active cells.
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Lab products found in correlation
Methyl thiazolyl tetrazolium (mtt), by Merck Group (6 mentions)
Enspire, by PerkinElmer (2 mentions)
Arvo mx model, by PerkinElmer (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Roche (1 mentions)
Prism 5, by GraphPad (1 mentions)
96 well microplate, by Thermo Fisher Scientific (1 mentions)
Wallac 1420 victor plate reader, by PerkinElmer (1 mentions)
Hanks balanced salt solution (hbss), by Merck Group (1 mentions)
96 well microplate, by Corning (1 mentions)
Victor nivo 3s, by PerkinElmer (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Meilun (1 mentions)
Victor x, by PerkinElmer (1 mentions)
Envision 2105, by PerkinElmer (1 mentions)
Prism 8, by GraphPad (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Solarbio (1 mentions)
9 protocols using methyl thiazolyl tetrazolium (mtt)
1
Antiproliferative Activity of Compounds
2
Evaluating Cell Growth Inhibition Strategies
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DLD-1 cells were seeded onto normal or poly-HEMA-coated flat bottom 96 well culture plates (1 × 104 per well) and treated with/without 25-hydroxycholesterol (25-HC, Sigma Aldrich) and/or difluoromethylornithine (DFMO, LKT laboratories or SCADS Inhibitor Kit, Screening Committee of Anticancer Drugs) and/or N-(3-Aminopropyl)cyclohexylamine (APCHA, Tokyo Chemical Industry, Japan) for 72 h. Cell growth was determined using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Roche) as described previously [15 (link)]. In brief, DLD-1 cells were treated with MTT for 4 h and dissolved with SDS overnight. The production of formazan due to reduction of MTT by living cells was measured at 570 nm using a spectrophotometer (ARVO MX model or Nivo, PerkinElmer).
3
MTT Assay for Belinostat and Cubisbel
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Cell viability was determined by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Cells were seeded in 96-well plates at a density of 1 × 104 cells/well and subsequently exposed to various concentrations of belinostat or Cubisbel for 24, 48 and 72 h. For synergy assays, cells were co-treated with varying concentrations of belinostat + CuCl2 ranging from 0.3 µM to 10 µM at a molar ratio ranging from 1:1 to 1:33 for each drug respectively for 72 h. Hence, 15 µl of 5 mg/ml MTT (Sigma Aldrich) prepared in phosphate-buffered saline (PBS) was added to each well and incubated for 3 h at 37°C in darkness. Following removal of MTT and addition of DMSO, absorbency was read on the Perkin Elmer Victor Wallace Multilabel Counter 1420 Microplate Reader at 570 nm. Percentage viability was determined by normalizing the averaged absorbencies of control cells to 100%. Drug IC50 values 72 h post-treatment were calculated using GraphPad Prism 5 software (GraphPad Software, Inc., USA v8.3.1) using the log(inhibitor) vs. normalized response—variable slope function.
4
Cytotoxicity Evaluation of Synthetic Compounds
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Cells were plated at a density of 1200 cells/well for HeLa, 1000 for A375, 10,000 for Jurkat and 2000 for WM266 and HDF in 96-well microplates (Thermofisher, Waltham, MA, USA). After 24 h incubation, cells were treated with increasing concentrations of synthetized compounds for 48 h. Cell proliferation was determined by using MTT (Sigma Aldrich, St. Louis, MO, USA) for adherent cells [62 (link)]. In the case of Jurkat that are suspension lymphoblasts, the proliferation was investigated by CCK-8 assay [63 (link)]. Plates were then analyzed by using a microplate reader (Enspire, Perkin Elmer, Waltham, MA, USA) at 570 nm for MTT and at 450 for CCK-8. The mean value of proliferating cells for each treatment was compared to untreated cells (control). All experiments were performed at least in triplicate and repeated at least 3 times. The IC50 values were calculated using Graph-Pad Prism (version 6.01, Graph-Pad software Inc., San Diego, CA, USA).
5
Colorimetric Viability Assay using MTT
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The number of viable cells was determined by the mitochondrial conversion of yellow MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to purple formazan dye. MTT (Sigma) was dissolved in Hank’s balanced salt solution (HBSS; Sigma), filtered, protected from light, and stored at 4 °C. HAPI cells were seeded on 96-well plates at a density of 15,000 cells per well. The next day, cells were treated with various concentrations of P. mirifica extract or LPS (100 ng/mL) and incubated for 24 h. The medium was removed and 10 μL of 10 mg/mL MTT in HBSS was added to each well. The cultures were incubated for 4 h in a humidified atmosphere at 37 °C and 5% CO2. Then, MTT was removed, cells were solubilized with 100 μL dimethyl sulfoxide, and the absorbance was measured at 570 nm on a microplate reader (Wallac 1420 Victor plate reader, Perkin-Elmer, Foster City, CA, USA). The results are shown as the percentage of the control (no treatment).
6
Cell Proliferation Assay Protocol
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Cells were seeded at density of 2000 cells/well for WM266, 1200 cells/well for HeLa, 1000 cells/well for A375 and 2000 cells/well for HDF in 96-well microplates (Corning), with 100 µL of medium for well. After 24 h incubation, cells were treated with the substances at the indicated concentrations. The compounds were solubilized in DMSO at 50 mM concentration. Cell proliferation was revealed after 48 h treatment using the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assay (MTT, Sigma Aldrich). Plates were then analyzed by using a microplate reader (Enspire, Perkin Elmer, Waltham, MA, USA) at 570 nm (MTT) [26 (link)]. The results are presented as the percentage of proliferating cells versus the control (DMSO treated cells) and are expressed as means ± SD of, at least, two independent experiments performed in triplicate. Statistical significance was determined by two-sided paired Student’s t-test. A p value less than 0.05 was considered significative. The IC50 values were calculated by GraphPad Prism software.
7
MTT Cytotoxicity Assay for Epithelial Cells
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A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed to determine the cytotoxic effects of transfection and superoxide on epithelial cell viability. MTT (Sigma-Aldrich) (100 μL of 100 μg/mL in DMEM) was added to wells on a 96-well plate and the plate returned to a tissue culture incubator for 2 h. The media/MTT mixture was aspirated and formazan crystals were dissolved in 200 μL methanol and absorbance read in a VICTOR™ X plate reader (Perkin Elmer, Waltham, MA) at 595 nm wavelength. Untreated cells were regarded as 100% viable as control (n = 3).
8
Cytotoxicity Assessment of CFTR Potentiators
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Cytotoxicity of CFTR potentiators was assessed by the conversion of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; Sigma-Aldrich/Merck, Saint-Quentin-Fallavier, France) into formazan crystals using living cells, as described [36 (link)]. Briefly, subconfluent MDCK cells were plated into 96-well plates in triplicate for each tested condition, including controls (no cells and 0.1% DMSO). Sixteen hours after cell seeding and 2 h after drug treatment, MTT (125 µg/mL, final concentration) was added in each well, and cells were reincubated at 37 °C for 2 h. Then, culture medium was gently washed out, and cells were lysed in 100 µL DMSO, and the absorbance at 540 nm was measured using a Wallac Victor3 multilabel plate reader (Perkin Elmer, Massy, France). Cell survival was calculated for each triplicate, and, after background subtraction, means were expressed as percentages of the mean of cells treated with the vehicle only.
9
Metformin and Cisplatin Cytotoxicity Assay
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Both TCam-2 and NTERA-2 cells were adhered in 96-well plates (3 × 103 cells) with standard culture medium for 24 h. Then cell medium was replaced with 100 μL fresh medium supplemented with different concentrations of metformin (1 mM, 5 mM, 10 mM, 15 mM, 20 mM). Standard medium served as a control. Following exposure to the solutions for different experimental time (24 h, 48 h and 72 h), 10 μL of MTT (Solarbio, China) were added in each well and were incubated for 4 h at 37℃ in dark. Then, we discarded the MTT and added 150 μL of dimethyl sulfoxide (DMSO) to detect the optical density (OD) at 490 nm with a microplate reader (EnVision 2105, PerkinElmer, England). In order to find the 50% inhibitory concentrations (IC50 values) of cisplatin monotherapy, combination therapy (metformin + Cisplatin) and sequential treatment (metformin − cisplatin), the Pearson Correlation coefficient was calculated for showing the correlation between viability of cells and therapy strategies. All the data were calculated by GraphPad Prism 8.
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