3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide

Manufactured by Promega

Sourced in United States

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide is a chemical compound commonly used as a colorimetric assay for assessing cell metabolic activity. It is a yellow tetrazolium salt that is reduced to a purple formazan product by metabolically active cells.

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Lab products found in correlation

Penicillin and streptomycin, by Thermo Fisher Scientific (1 mentions) A dmem, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Trypan blue, by Thermo Fisher Scientific (1 mentions) G3581, by Promega (1 mentions) Pepstatin a, by Enzo Life Sciences (1 mentions) Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Hek293 cell, by American Type Culture Collection (1 mentions)

5 protocols using 3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide

Cytotoxicity Assays for MDA-MB-231 Cells

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MDA-MB-231 cells were seeded in 96-well plates (12,000 cells/well). After 24 h incubation, the cells were treated with different concentrations of compound 1–3, pepstatin A (4, Enzo Life Sciences), or solvent control (DMSO). Following 48 h of incubation, cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide according to the manufacturer’s instructions (Promega).
To measure cell viability using the same conditions as preparation of CM, MDA-MB-231 cells were seeded and after 24 h incubation, the media was replaced with serum free media (DMEM, without sodium bicarbonate, buffered with 50 mM HEPES, pH 6.6 adjusted using 1N HCl) and treated with compounds 1–3, pepstatin A (4), E-64, or solvent control. Cell viability was measured after 72 h using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide according to the manufacturer’s instructions (Promega).

Al-Awadhi F.H., Law B.K., Paul V.J, & Luesch H. (2017). Grassystatins D–F, Potent Aspartic Protease Inhibitors from Marine Cyanobacteria as Potential Antimetastatic Agents Targeting Invasive Breast Cancer. Journal of natural products, 80(11), 2969-2986.

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Evaluating Genotoxic Stress Response in Fibroblasts

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Fibroblasts were isolated from skin biopsies of 6 healthy controls and 6 patients carrying the identified APTX mutation. Cells were cultured in Advanced DMEM/F-12 (Dulbecco's Modified Eagle Medium/Ham's F-12) (ADMEM, Gibco) containing 10% fetal bovine serum (FBS) (Gibco), 2mM L-glutamine (Life Technologies), 100 U/ml penicillin and streptomycin (Gibco) and maintained in a humidified incubator under 37°C and 5% CO₂. Cells were cultured in 96-well plates at a density of 5000 cells/well. The following day, cells were treated in triplicates with increasing concentrations of mitomycin-C (MMC), methyl-methane sulfonate (MMS) and etoposide for 1 hour, and H₂O₂ for 30 minutes, under 37°C and 5% CO₂. Cells in normal culture media with no treatment served as a control. To assess the effect of these different genotoxic agents on cell survival, we performed MTT assay [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] (Promega, USA) after 48 hours of treatment and the percentage of viable cells was calculated accordingly.

Ababneh N.A., Ali D., Al-Kurdi B., Sallam M., Alzibdeh A.M., Salah B., Ryalat A.T., Azab B., Sharrack B, & Awidi A. (2020). Identification of APTX disease-causing mutation in two unrelated Jordanian families with cerebellar ataxia and sensitivity to DNA damaging agents. PLoS ONE, 15(8), e0236808.

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Cell Viability Assessment Protocols

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For MTS assays, cells were seeded in a 96-well plate with densities of 1 × 10⁴ cells/well (AU565), 1 × 10⁴ cells/well (SKBR3), and 5 × 10³ cells/well (HCC-1954). After 24 h, cells were treated with desired concentrations of the ligands and incubated for 72 h without changing media. After 72 h, NADH-dependent tetrazolium bromide reduction was measured using an MTS assay as an indirect indication of the viable cell quantity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Promega, Madison, WI, USA #G3581). A Trypan Blue exclusion assay was performed to determine viable cell count. Cells were seeded in 6-well plates in different densities—2 × 10⁵ cells/well (AU565), 2 × 10⁵ cells/well (SKBR3), and 1 × 10⁵ cells/well (HCC-1954). After 24 h, the plates were treated with DMSO (vehicle), GW3965 (10 μM), 1E5 (10 μM), and incubated for 72 h. Viable cells were then counted using a hemocytometer upon staining with 0.4% Trypan Blue (Gibco, 15250061).

Premaratne A., Basu S., Bagchi A., Zhou T., Feng Q, & Lin C.Y. (2024). Liver X Receptor Ligand GAC0001E5 Downregulates Antioxidant Capacity and ERBB2/HER2 Expression in HER2-Positive Breast Cancer Cells. Cancers, 16(9), 1651.

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HEK293 Cell Cytotoxicity Assay

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HEK293 cells were purchased from ATCC (Manassas, VA, USA). They were cultured and maintained in Dulbecco’s modified Eagle medium (DMEM; Invitrogen, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA) and 1% antibiotic-antimycotic (Invitrogen) at 37 °C in a humidified atmosphere with 5% CO₂. HEK293 cells were seeded in 96-well plates at densities of 15,000 cells per well in 100 μL, respectively. After 16 h of incubation, the cells were treated with 0.5 μL of various concentrations of compounds or a DMSO solvent control. Following 24 h of incubation, cells were treated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide according to the manufacturer’s instructions (Promega, Madison, WI, USA). The experiments were performed as technical triplicates.

Elsadek L.A., Ellis E.K., Seabra G., Paul V.J, & Luesch H. (2023). Chlorinated Enyne Fatty Acid Amides from a Marine Cyanobacterium: Discovery of Taveuniamides L-M and Pharmacological Characterization of Taveuniamide F as a GPCR Antagonist with CNR1 Selectivity. Marine Drugs, 22(1), 28.

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MTT Assay for Cell Viability

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MCF-7 cells were seeded in 96-well plate, incubated with increasing concentrations (from 2.5 to 150 µg.mL -1 ) of the different isomers for 3 days and the percentage of living cells was quantified by MTT assay. This consists in a colorimetric test performed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Promega) where cells were incubated with 0.5 mg•mL -1 MTT for 4 h to determine mitochondrial enzyme activity. Then, MTT precipitates were dissolved in 150 µL ethanol/DMSO solution (in proportion 1/1) followed by 20 min of shaking and absorbance was read at 540 nm. Cells treated with the vehicle were served as control.

Rouillon J., Ali L.M.A., Hadj-Kaddour K., Marie-Luce R., Simon G., Onofre M., Denis-Quanquin S., Jean M., Albalat M., Vanthuyne N., Micouin G., Banyasz A., Gary-Bobo M., Monnereau C, & Andraud C. (2022). Assembly of Aggregation-Induced Emission Active Bola-Amphiphilic Macromolecules into Luminescent Nanoparticles Optimized for Two-Photon Microscopy In Vivo. Biomacromolecules, 23(6).

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