Methyl thiazolyl tetrazolium (mtt)

Manufactured by MP Biomedicals

Sourced in United States, France, China

MTT is a colorimetric assay used to measure the metabolic activity of cells. It is a salt that is reduced by the mitochondria of living cells, resulting in the formation of a purple formazan product that can be quantified spectrophotometrically.

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Lab products found in correlation

Dimethyl sulfoxide (dmso), by MP Biomedicals (8 mentions) Fluostar omega plate reader, by BMG Labtech (4 mentions) Microplate reader, by Bio-Rad (3 mentions) Varioskan flash multimode reader, by Thermo Fisher Scientific (2 mentions) Wst 1 reagent, by Roche (2 mentions) Fingolimod, by Cayman Chemical (2 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Sphingosine 1 phosphate (s1p), by Avanti Polar Lipids (2 mentions) Microplate reader, by Thermo Fisher Scientific (2 mentions) Elx800, by Agilent Technologies (1 mentions) Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions) 96 well cell culture plate, by Corning (1 mentions) Verapamil, by MP Biomedicals (1 mentions) Paclitaxel, by MP Biomedicals (1 mentions) Doxorubicin, by MP Biomedicals (1 mentions) Spectramax i3x spectrophotometer, by Molecular Devices (1 mentions) Second antibody, by Santa Cruz Biotechnology (1 mentions) Trypan blue, by Welgene (1 mentions) Sb203580, by Merck Group (1 mentions)

37 protocols using methyl thiazolyl tetrazolium (mtt)

Cytotoxicity Assay of MDA-MB-231 Cells

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MDA-MB-231 breast cancer cell lines were purchased from the National Centre for Cell Science (NCCS), Pune, India. MDA-MB-231 cells were grown in L15 media with 10% FBS (fetal bovine serum), 100 IU/ml penicillin, 100 g/ml streptomycin, and 0.25 µg/ml amphotericin B at 37°C in a humid incubator with 5% CO 2 .
Cell viability assay was performed, and 1x10 4 MDA-MB-231 cells were seeded per well in 96-well plates. After 24 hours of incubation in a 5% CO 2 incubator at 37°C, the cells were treated with different concentrations (10µg/ml, 50 µg/ml, 100 µg/ml and 200 µg/ml) of the prepared extracts. MTT assay was performed as described earlier [24] (link), in brief after 48 hours of incubation, 20 µL of the MTT (MP Biomedicals, USA) stock solution (5mg/ml) was added to each well, followed by additional 4 hours of incubation. After which, 100µl of the solubilizing buffer was added to each well to dissolve the formazan crystals. The absorbance was taken at 570 nm wavelength using an Elisa reader (Epoch, Biotek, USA). Further, the percentage inhibition was calculated by comparing the percentage of viability with the untreated control. The experiment was performed in triplicates to verify the results. Paclitaxel was taken as a positive control.

Upreti S., Muduli K., Pradhan J., Elangovan S, & Samant M. (2023). Identification of novel inhibitors from Urtica spp against TNBC targeting JAK2 receptor for breast cancer therapy. Medical oncology (Northwood, London, England), 40(11).

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Luteal Cell Proliferation Assay

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The wild type luteal cells and EGR 1 KO luteal cell were seeded on 96-well plates and incubated at 37 °C for 72 hr with media without treatment and with media containing VEGF A and FGF 2 (100, 50 ng mL⁻¹). Subsequently 20 µl of MTT solution (5 mg/ml in PBS) 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltertrazolium bromide (MTT; MP Biomedical) was added to each well and was incubated for 4 hr at 37 °C. Supernatant was then removed and 100 mL dimethyl sulphoxide (DMSO; MP Biomedical) was added and absorbance at 450 nm was detected with a microplate reader (Biorad).

Punetha M., Chouhan V.S., Sonwane A., Singh G., Bag S., Green J.A., Whitworth K, & Sarkar M. (2020). Early growth response gene mediates in VEGF and FGF signaling as dissected by CRISPR in corpus luteum of water buffalo. Scientific Reports, 10, 6849.

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Cell Viability Assay for shRNA Transfected MCF7 Cells

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For cell viability assay, shRNA transfected MCF7 cells were splitted into 24 well plate at a density of 2x10⁵ cells per well. Next day, the medium was changed to fresh medium containing puromycin (1 μg/ml). Cell viabilities were determined by MTT assay after 24 and 48 h of puromycin treatment. For MTT assay, 25 μl of 5 mg/ml MTT (MP Biomedicals) solution was added to each well and incubated for 4 h at 37°C. The formazan crystals were dissolved in DMSO and analyzed using a microplate reader (Varioskan Flash, Thermo Scientific, USA) at 570 nm with background subtraction at 630 nm.

Mishra D.R., Chaudhary S., Krishna B.M, & Mishra S.K. (2015). Identification of Critical Elements for Regulation of Inorganic Pyrophosphatase (PPA1) in MCF7 Breast Cancer Cells. PLoS ONE, 10(4), e0124864.

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Sanguinarine Cytotoxicity Assay

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Cells were seeded in 96 wells plates at density of 0.8 × 10⁴ cells per well. Sanguinarine was dissolved in DMSO as a stock solution and was stored in aliquots at − 20 °C. After treatment with or without sanguinarine, MTT (3-[4, 5-dimethylthiazolyl-2]-2, 5-diphenyl tetrazolium bromide) (MP, France) was added into each well. Cells were incubated for 4 h. The formazan crystals were dissolved in DMSO. The absorbance value was measured at 490 nm.

Gong X., Chen Z., Han Q., Chen C., Jing L., Liu Y., Zhao L., Yao X, & Sun X. (2018). Sanguinarine triggers intrinsic apoptosis to suppress colorectal cancer growth through disassociation between STRAP and MELK. BMC Cancer, 18, 578.

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Cytotoxicity Assay of Doxorubicin and Paclitaxel

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Tumor cells were plated in triplicate at a density of 5×10³ cells/well in a 96 well plate and incubated for 24 hrs at 37°C. Cells were treated with different concentrations of doxorubicin or paclitaxel for 72 hrs. After 72 hrs, supernatants were collected from each well and fresh media was added to the cells along with 30μL of 5mg/ml MTT (3-(4, 5-Dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide) (MP Biomedicals) and incubated for 2 hrs. After incubation, the media was aspirated and the precipitates were dissolved in 100 μL of DMSO (Fisher Scientific) with shaking. Absorbance was read at 570 nm on an ELx800 Bio-Tek plate reader. Percent inhibition was calculated using the following formula [100-(Absorbance of treatment / Absorbance of control group) ×100].

Sharma B., Varney M.L., Saxena S., Wu L, & Singh R.K. (2016). Induction of CXCR2 ligands, stem cell-like phenotype, and metastasis in chemotherapy-resistant breast cancer cells. Cancer letters, 372(2), 192-200.

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Cell Viability Assay in Breast Cancer

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Cell viability assay was conducted as previously described [40 (link)]. Breast cancer control, GPR141 overexpressed and knockdown cells were seeded in 96-well cell culture plates (Corning, Pune, India) at 3 × 10³ cells/well density. After 48 h incubation, 10 μL of MTT (MP Biomedical) (5 mg/mL in PBS) was added to each well and incubated at 37°C, 5% CO2 atmospheric condition for another 3 h. Then the medium was removed, and 100 μL of DMSO was added to dissolve the formed formazan crystals. The solubilized crystals were quantified by scanning the plates at 570 nm using Varioskan™ Flash Multimode Reader (Thermo Fisher Scientific, Bangalore, India).

Parija M., Adhya A.K, & Mishra S.K. (2023). G-protein-coupled receptor 141 mediates breast cancer proliferation and metastasis by regulating oncogenic mediators and the p-mTOR/p53 axis. Oncotarget, 14, 466-480.

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Cell Viability Assay Protocol

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Cell viability was measured using the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide assay (MTT; MP Biomedicals, Eindhoven, The Netherlands). Twenty-four hours post-transfection with the antagomirs, cells were seeded in 96-wells culture plates for the MTT assay. Simultaneously, RNA was extracted from a subset of the cells to determine the miRNA expression levels. MTT measurements were performed at t = 0 and subsequently after 3 and 6 days. The relative viability was determined by subtracting the measurement at day 0 from subsequent time points. All measurements were performed in triplicate (technical replicates) and at least 2 independent experiments were performed (biological replicates). Relative viability of cells was compared using Student’s t-test, with p-values <0.05 considered significant.

de Groen F.L., Timmer L.M., Menezes R.X., Diosdado B., Hooijberg E., Meijer G.A., Steenbergen R.D, & Carvalho B. (2015). Oncogenic Role of miR-15a-3p in 13q Amplicon-Driven Colorectal Adenoma-to-Carcinoma Progression. PLoS ONE, 10(7), e0132495.

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Cellular Proliferation Measurement Protocols

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Cellular proliferation was measured by MTT and cell count assay. Briefly, for MTT assay, 1×10⁴ cells were seeded into a 96-well plate and incubated for 1, 2, 3 and 4 days. Twenty microliters of MTT (5 mg/mL; MP Biomedicals, Santa Ana, CA, USA) was added to each well and incubated for 4 h. The absorbance value (OD) of each well was measured at 570 nm. For cell count assay, 2×10⁵ cells from each group were plated into six-well culture plates in complete culture medium for 0, 1, 2, 3 and 4 days. Then, the cell number was determined in triplicate using a hemocytometer.

Peng J., Chen W., Chen J., Yuan Y., Zhang J, & He Y. (2018). Overexpression of chloride channel-3 predicts unfavorable prognosis and promotes cellular invasion in gastric cancer. Cancer Management and Research, 10, 1163-1175.

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Anti-proliferation Measurement by MTT Assay

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The anti-proliferation property was measured by measuring cellular viabilities using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) purchased from MP Biomedicals, France. The resulting optical density (OD) of the formazan product produced by the MTT and living cells was measured at 570 nm. The OD of the non-treated group was defined as 100% proliferation, and the proliferation index of various treated groups was counted as: (OD of treated group / OD of paired non-treated group) × 100%.

Dachlan I., Wirohadidjojo Y.W., Wahyuningsih M.S., Aryandono T., Soebono H, & Afandy D. (2019). The effect of 5α-oleandrin on keloid fibroblast activities. BMC Proceedings, 13(Suppl 11), 14.

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Cytotoxicity Assay for Drug Resistance

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Paclitaxel, doxorubicin, verapamil, and MTT 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from MP Biomedicals (Santa Ana, CA). P-gp inhibitors identified though in silico screening were purchased in small quantities through SIA MolPort (Riga, Latvia). Stock solutions (10–100 mmol/L) of all drugs and experimental compounds were prepared in DMSO and stored as aliquots at −20°C. On the day of the experiment, working solutions of compounds prepared in DMSO were further diluted in culture media such that the final DMSO concentration was ≤1% (v/v). MTT solution was prepared as 5 mg/mL in phosphate buffered saline (PBS; 137 mmol/L NaCl, 2.7 mmol/L KCl, 10 mmol/L Na₂HPO₄, 1.8 mmol/L KH₂PO₄) and sterile filtered through 0.22 μm nylon filter before use. Cell culture materials were purchased from Corning Inc. (Corning, NY) unless otherwise stated. GraphPad Prism∼ version 6.05 for Windows was used for plotting data and IC₅₀ values were calculated from nonlinear, four-parameter logistic curve fitting (GraphPad Software, La Jolla, CA).

Follit C.A., Brewer F.K., Wise J.G, & Vogel P.D. (2015). In silico identified targeted inhibitors of P-glycoprotein overcome multidrug resistance in human cancer cells in culture. Pharmacology Research & Perspectives, 3(5), e00170.

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