Mtt method

Manufactured by Merck Group

Sourced in United States

The MTT method is a colorimetric assay used to measure the metabolic activity of cells. It is a widely-used technique in cell biology and biochemistry research.

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Lab products found in correlation

Clone tdm29, by Merck Group (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Venor gem, by Minerva Biolabs (1 mentions) Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions) Wallac victor2 1420 multilabel counter, by PerkinElmer (1 mentions) Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions) Microplate reader, by Agilent Technologies (1 mentions) Cell titer blue h cell viability assay kit, by Promega (1 mentions) Microplate reader, by Bio-Rad (1 mentions)

Close spelling variants of this product

MTT colorimetric method

10 protocols using mtt method

Evaluating Breast Cancer Cell Viability

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Viability of human and mouse breast cancer cell lines was evaluated by using the MTT method, according to the manufacturer’s protocol (Sigma-Aldrich).
To quantify the effect of conditioned media (CM) from empty and HBB transfected tumour cells on HUVEC cell density, the colorimetric crystal violet method was employed. Cells were serum starved for 6 h, then medium was replaced with EGM-2 medium with or without CM from MDA-empty and MDA-HBB. After 24 h, cells were fixed with 3% formaldehyde plus 2% sucrose solution and stained with 5% crystal violet in 20% methanol. Stain was then dissolved with 0.1 M sodium citrate (pH 4.2) and the relative absorbance evaluated at 595 nm.

Ponzetti M., Capulli M., Angelucci A., Ventura L., Monache S.D., Mercurio C., Calgani A., Sanità P., Teti A, & Rucci N. (2017). Non-conventional role of haemoglobin beta in breast malignancy. British Journal of Cancer, 117(7), 994-1006.

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Cell Adhesion Assay for Integrin Subunits

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Cell adhesion assays were performed as previously described [30] (link). 96-well microplates were coated with a solution of 10 µg/ml type 1 collagen from calf skin (Sigma-Aldrich; MO, USA) in PBS, or PBS-1% BSA. For each well, 2.5×10⁴ cells were seeded and incubated at 37°C for 20 min. In selected experiments, cells were previously incubated with function-blocking mAbs against integrin subunits α2 (clone P1E6, Chemicon; 1/50 in PBS), α3 (clone P1B5, Calbiochem, CA, USA; 1/50), α5 (clone P1D6, Chemicon; 1/50) or β1 (clone TDM29, Chemicon; 1/5) for 30 min at 4°C. After three washes, adherent cells were estimated with the MTT method (Sigma). Three independent experiments were performed in quadruplicate. Results were expressed as the mean ± standard deviation (SD) of values of specific binding to type 1 collagen (OD 570 nm of cells bound – OD 570 nm of cells bound to PBS-1% BSA).

Bassagañas S., Carvalho S., Dias A.M., Pérez-Garay M., Ortiz M.R., Figueras J., Reis C.A., Pinho S.S, & Peracaula R. (2014). Pancreatic Cancer Cell Glycosylation Regulates Cell Adhesion and Invasion through the Modulation of α2β1 Integrin and E-Cadherin Function. PLoS ONE, 9(5), e98595.

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SJAMP Modulates SW1990 Proliferation

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SW1990 proliferation was evaluated by MTT method (Sigma-Aldrich, USA). After affected by SJAMP of 0.5, 1, 2, 4, 8 mg/ml for 0, 24, 48, 72 hs, SW1990 were seeded into 96-well plates with 200 μl medium and 1 × 10⁴ cells per well. Changing fresh culture medium with MTT (5 mg/ml in PBS, 200 μl per well) at each time point, and then continue to incubate for another 4 h [43 (link)]. Following incubation, medium was discarded and dimethyl sulfoxide (DMSO 150 μl/well; Sigma-Aldrich) was added to each well for 10 mins. Absorbance at 490 nm is analyzed by an automated reader [43 (link)] (Bio-Rad, Hercules, CA, USA). To determine the effect of SJAMP to cell proliferation after treatment with YAP siRNA, the same procedures were performed to SW1990 with 8 mg/ml SJAMP for 24 h, 48 h, 72 h, after transfected with YAP siRNA.

Li X., Liu Y., Zhang C., Niu Q., Wang H., Che C., Xie M., Zhou B., Xu Y., Zhang Q., Wu J, & Tian Z. (2017). Stiehopus japonieus acidic mucopolysaccharide inhibits the proliferation of pancreatic cancer SW1990 cells through Hippo-YAP pathway. Oncotarget, 8(10), 16356-16366.

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Cell Line Authentication and Cytotoxicity Assay

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Cell lines were obtained from the American Type Culture Collection and grown in their recommended culture medium, supplemented with 10% FBS at 37 °C in 5% CO₂. In-house authentication of cell lines by SNP profiling was carried out and cultured cells were passaged for less than 6 months before replacement from early-passage frozen stocks. Cells were regularly screened for Mycoplasma, using a PCR-based assay (VenorGem; Minerva Biolabs, Berlin, Germany). Transfections were carried out at ∼80% confluency with the plasmids indicated, using Lipofectamine LTX (15338030, Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. Cell proliferation assays were carried out by colorimetric MTT method (Sigma, St Louis, MO, USA) as described elsewhere (Mosmann, 1983 (link)). Briefly, cells were plated in 96-well plates at 2000–5000 cells per well (depending on the cell line) followed by treatment with two-fold dilutions of 0–25 μM drug for 72 h. Absorbance was measured at 570 nm with the Wallac VICTOR2 1420 Multilabel Counter (PerkinElmer, Waltham, MA, USA).

Faisal A., Mak G.W., Gurden M.D., Xavier C.P., Anderhub S.J., Innocenti P., Westwood I.M., Naud S., Hayes A., Box G., Valenti M.R., De Haven Brandon A.K., O'Fee L., Schmitt J., Woodward H.L., Burke R., vanMontfort R.L., Blagg J., Raynaud F.I., Eccles S.A., Hoelder S, & Linardopoulos S. (2017). Characterisation of CCT271850, a selective, oral and potent MPS1 inhibitor, used to directly measure in vivo MPS1 inhibition vs therapeutic efficacy. British Journal of Cancer, 116(9), 1166-1176.

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Spleen Lymphocyte Proliferation Assay

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Two weeks after the final immunization, to euthanize mice, they were first anesthetized with ketamine (75 mg/kg) injected subcutaneously. The spleen of each mouse was removed, and lymphocyte proliferation was evaluated using the MTT method according to the manufacturer´s recommendations (Sigma) by Phytohemagglutinin (PHA) and TC1 lysate. In this way, we prepared the TC1 cell by sonicating and removing the cell wall and releasing the protein.

Sadr-Momtaz S., Aftabi M., Behboudi E., Naderi M., Hashemzadeh-Omran A, & Moradi A. (2023). NSP4 as adjuvant for immunogenicity and design of effective therapeutic HPV16 E6/E7/L1 DNA vaccine in tumor-bearing and healthy C57BL/6 mice. BMC Research Notes, 16, 164.

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MTT Proliferation Assay for Gastric Cancer

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MTT method (Sigma, Shanghai) was applied for proliferation detection of GC cells. Cells were seeded into 96-well plates at a density of 2 × 10³ cells/well. After 24 h, 48 and 72 h, respectively, 20 µL of MTT (0.5 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) was added into each well for incubation at 37 ℃ for 4 h. The precipitate was solubilized in 150 µL of RPMI-1640. The OD values at 570 nm were recorded. The experiment was repeated three times.

Zhou K., Song B., Wei M., Fang J, & Xu Y. (2020). MiR-145-5p suppresses the proliferation, migration and invasion of gastric cancer epithelial cells via the ANGPT2/NOD_LIKE_RECEPTOR axis. Cancer Cell International, 20, 416.

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Cell Viability Assay of RNase

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Cells were seeded into 96-well plates at 1500 cells/well for OVCAR-8, and at 1900 for NCI-H460. After 24 h of incubation, cells were treated with various concentrations of RNase for 72 h. Drug sensitivity was determined by the MTT method essentially following the manufacturer’s instructions (Sigma, Saint Louis, MO, USA) and according to [39 (link)]. All data are presented as the mean ± SE of at least three independent experiments with three replicates for each.

Roura Padrosa D., Castro J., Romero-Casañas A., Ribó M., Vilanova M, & Benito A. (2018). Construction of Highly Stable Cytotoxic Nuclear-Directed Ribonucleases. Molecules, 23(12), 3273.

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Evaluating Cytotoxicity of Plasma Exosomes on HepG2 Cells

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The cytotoxicity of plasma exosomes against HepG2 cells was evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) method (Sigma). Briefly, the cells were seeded in 96-well plates and treated with the doses of 0.25–64 µg/mL the pooled exosomes obtained from obese women (PO-Exo). After 24 h of incubation at 37 °C, the media were removed and 100 μL MTT solution (0.5 mg/mL in PBS) was added to each well and incubated at 37 °C for 4 h. After removing the MTT solution, 100 μL dimethyl sulfoxide (DMSO) was added to each well and the plate was shaken in dark for 10 min. Finally, the absorbance was determined at 570 nm using the microplate reader (BioTek, USA). After the determination of the dose, the MTT assay was again used to evaluating the effect of plasma exosomes derived from normal-weight and obese females on cell viability.

Afrisham R., Sadegh-Nejadi S., Meshkani R., Emamgholipour S, & Paknejad M. (2020). Effect of circulating exosomes derived from normal-weight and obese women on gluconeogenesis, glycogenesis, lipogenesis and secretion of FGF21 and fetuin A in HepG2 cells. Diabetology & Metabolic Syndrome, 12, 32.

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Cell Proliferation Assay Protocol

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Cell proliferation was evaluated using the MTT method (Sigma-Aldrich, St. Louis, MO, USA) and Cell Titer-Blue H^® Cell Viability Assay Kit (Promega). After treatment, the cells were incubated for 0, 24, 48, and 72 h before adding the MTT reagent to each well at a final concentration of 0.5 mg/ml and incubated at 37°C for 4 h. After medium removal, 500 µl of dimethyl sulfoxide was added to each well. Viable cells were measured by absorbance at a 550 nm wavelength using a microplate reader (Bio-Rad, Hercules, CA, USA). Cell Titer-Blue H^® Cell Viability Assay was carried out according to the manufacturer’s instructions.

Ma Y., Feng J., Xing X., Zhou B., Li S., Zhang W., Jiang J., Zhang J., Qiao Z., Sun L., Ma Z, & Kong R. (2016). miR-1908 Overexpression Inhibits Proliferation, Changing Akt Activity and p53 Expression in Hypoxic NSCLC Cells. Oncology Research, 24(1), 9-15.

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Evaluating S. aureus and MG Effects on RAW264.7 Cells

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The effect of S. aureus and MG on RAW264.7 cells viability was assayed using MTT method (Sigma-Aldrich, America). Cells were inoculated into a 96-well plate at 2×10⁴ CFU/well, after cells growing to monolayer, washing three times with phosphate buffer saline (PBS; Hyclone; China). Then, 200 µL of S. aureus (MOI = 100:1) was added into each well for different times (0, 0.5, 1, 2, 4, and 6 h) (three wells/each group), or 200 µL of MG of different concentrations to each well (three wells each concentration). After washing three times with PBS, the cells were incubated with color-free DMEM (80 µL/well) and 5 mg/mL MTT (20 µL/well) solution at 37°C for 4 h. After washing three times with PBS, 0.05% DMSO (100 µL/well) was added and the tray was shaken gently for 10 min. The cells were observed under a light microscope. The optical density (OD) value of the wells at a wavelength of 490 nm was measured using an enzyme-inked immunosorbent assay reader (Thermo Fisher Scientific, US).29 (link) The percentage of viable cells was calculated according to the equation: the percentage of viable cells = (OD-value of test team/OD-value of a blank team) ×100%.30 (link)

Xu J., Yao H., Wang S., Li H, & Hou X. (2020). Mangiferin Inhibits Apoptosis and Autophagy Induced by Staphylococcus aureus in RAW264.7 Cells. Journal of Inflammation Research, 13, 847-857.

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