3 4 5 dimethylthiazol 2 yl 5 3 carboxymethoxyphenyl 2 4 sulfophenyl 2h tetrazolium mts

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3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) is a tetrazolium compound used in colorimetric assays to measure cell viability and proliferation. It functions as a substrate for cellular dehydrogenase enzymes, which reduce the MTS compound to formazan, producing a colored product that can be quantified spectrophotometrically.

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43 protocols using 3 4 5 dimethylthiazol 2 yl 5 3 carboxymethoxyphenyl 2 4 sulfophenyl 2h tetrazolium mts

In Vitro Cytotoxicity Assessment of Compounds

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Compounds were assayed at concentrations of 5 μg/mL, 10 μg/mL, 20 μg/mL, and 40 μg/mL against a murine macrophage cell line (J774.A1) (ATCC TIB-67, American Type Culture Collection (ATCC), Manassas, VA, USA) to determine the potential toxic effect in vitro. Cells were cultured in Dulbeco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO, USA) with 10% fetal bovine serum (USA Scientific, Inc.) at 37°C with 5% CO₂. Controls received DMSO alone at a concentration equal to that in drug-treated cell samples. The cells were incubated with the compounds in a 96-well plate at 37°C and 5.0% CO₂ for two hours prior to addition of the assay reagent MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (Promega, Madison, WI, USA). Absorbance readings were taken using a kinetic ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA). The quantity of viable cells after treatment with each compound was expressed as a percentage of the control, DMSO.

Mohammad H., Reddy P.V., Monteleone D., Mayhoub A.S., Cushman M., Hammac G.K, & Seleem M.N. (2015). Antibacterial Characterization of Novel Synthetic Thiazole Compounds against Methicillin-Resistant Staphylococcus pseudintermedius. PLoS ONE, 10(6), e0130385.

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Compound 35 Cytotoxicity Evaluation

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Compound 35 was evaluated (at concentrations ranging from 16 to 128 μM) against a human keratinocyte (HaCaT) cell line (AddexBio, San Diego, CA, USA) to determine the potential toxic effect to mammalian skin cells in vitro, as described in a previous report [23 (link)]. In brief, keratinocytes were cultured in DMEM supplemented with 10% FBS at 37 °C with CO₂ (5%). Control cells received DMSO alone at a concentration equal to that in compound-treated cell samples. Cells were incubated with compound 35 (using triplicate samples) in a 96-well plate at 37 °C with CO₂ (5%) for 24 hours. The assay reagent MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (Promega, Madison, WI, USA) was subsequently added and the plate was incubated for four hours. Absorbance readings (at OD₄₉₀) were taken using a kinetic microplate reader (Molecular Devices, Sunnyvale, CA, USA). The quantity of viable cells after treatment with compound 35 was expressed as a percentage of the viability of DMSO-treated control cells (average of triplicate wells ± standard deviation). The toxicity data was analyzed via a one-way ANOVA, with post hoc Dunnet’s multiple comparisons test (P < 0.05), utilizing GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA).

Mohammad H., Kyei-Baffour K., Abutaleb N.S., Dai M, & Seleem M.N. (2019). An aryl isonitrile compound with an improved physicochemical profile that is effective in two mouse models of multidrug-resistant Staphylococcus aureus infection. Journal of global antimicrobial resistance, 19, 1-7.

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In vitro Mammalian Skin Cell Toxicity

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Compounds 7, 18, and 19 were assayed (at concentrations of 16, 32, 64, and 128 µM) against a human keratinocyte (HaCaT) cell line (AddexBio, San Diego, CA, USA) to determine the potential toxic effect to mammalian skin cells in vitro, as previously described.^{44 (link)–48 (link)} In brief, cells were cultured in DMEM supplemented with 10% FBS at 37 °C with CO₂ (5%). Control cells received DMSO alone at a concentration equal to that in drug-treated cell samples. The cells were incubated with the compounds (in triplicate) in a 96-well plate at 37 °C with CO₂ (5%) for 24 hours. The assay reagent MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (Promega, Madison, WI, USA) was subsequently added and the plate was incubated for four hours. Absorbance readings (at OD₄₉₀) were taken using a kinetic microplate reader (Molecular Devices, Sunnyvale, CA, USA). The quantity of viable cells after treatment with each compound was expressed as a percentage of the viability of DMSO-treated control cells (average of triplicate wells ± standard deviation). The toxicity data was analyzed via a one-way ANOVA, with post hoc Dunnet’s multiple comparisons test (P < 0.05), utilizing GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA).

Kyei-Baffour K., Mohammad H., Seleem M.N, & Dai M. (2019). Second-generation aryl isonitrile compounds targeting multidrug-resistant Staphylococcus aureus. Bioorganic & medicinal chemistry, 27(9), 1845-1854.

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Cytotoxicity Screening of Compounds

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Compounds 1–5 were assayed (at concentrations of 5 μg/mL, 10 μg/mL, 20 μg/mL, and 40 μg/mL) against a human keratinocyte (HaCaT) cell line (Catalogue Number: T0020001, AddexBio, San Diego, CA, USA) to determine the potential toxic effect to mammalian skin cells in vitro as described before [18 (link)]. Briefly, cells were cultured in DMEM supplemented with 10% FBS at 37°C with CO₂ (5%). Control cells received DMSO alone at a concentration equal to that in drug-treated cell samples. The cells were incubated with the compounds (in triplicate) in a 96-well plate at 37°C with CO₂ (5%) for two hours prior to addition of the assay reagent MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (Promega, Madison, WI, USA). Absorbance readings (at OD₄₉₀) were taken using a kinetic microplate reader (Molecular Devices, Sunnyvale, CA, USA). The quantity of viable cells after treatment with each compound was expressed as a percentage of the viability of DMSO-treated control cells (average of triplicate wells ± standard deviation). The toxicity data was analyzed via a one-way ANOVA, with post hoc Dunnet’s multiple comparisons test (P < 0.05), utilizing GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA).

Mohammad H., Cushman M, & Seleem M.N. (2015). Antibacterial Evaluation of Synthetic Thiazole Compounds In Vitro and In Vivo in a Methicillin-Resistant Staphylococcus aureus (MRSA) Skin Infection Mouse Model. PLoS ONE, 10(11), e0142321.

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Cytotoxicity Evaluation of Phenylthiazole Compounds

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Phenylthiazole compounds were assayed (at concentrations of 8, 16, and 32) against a human colorectal adenocarcinoma (Caco-2) cell line to determine the potential toxic effect to mammalian cells in vitro. Briefly, cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), non-essential amino acids (1X), penicillin-streptomycin at 37 °C with 5% CO₂. Compounds were added and serially diluted. Control cells received DMSO (the solvent of the compounds) alone at a concentration equal to that in compound-treated wells to determine the baseline measure of the cytotoxic impact of the compounds. The cells were incubated with the compounds (in triplicate) in a 96-well plate at 37 °C with 5% CO₂ for 24 hours. The assay reagent MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (Promega, Madison, WI, USA) was subsequently added and the plate was incubated for four hours. Absorbance readings (at OD₄₉₀) were recorded using a kinetic microplate reader (Molecular Devices, Sunnyvale, CA, USA). The quantity of viable cells after treatment with each compound was expressed as a percentage of the viability of DMSO-treated control cells (average of triplicate wells ± standard deviation).

Omara M., Hagras M., Elsebaie M.M., Abutaleb N.S., Nour El-Din H.T., Mekhail M.O., Attia A.S., Seleem M.N., Sarg M.T, & Mayhoub A.S. (2023). Exploring novel aryl/heteroaryl-isosteres of phenylthiazole against multidrug-resistant bacteria. RSC Advances, 13(29), 19695-19709.

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Cytotoxicity Evaluation of Compounds

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Compounds were assayed (at concentrations of 32, 64, 128, and 256 μM) against a human colorectal (HRT-18) cell line to determine the potential toxic effect to mammalian cells in vitro. Briefly, cells were cultured in RPMI-1640 medium supplemented with 10% fetal horse serum at 37 °C with CO₂ (5%). Control cells received DMSO alone at a concentration equal to that in drug-treated cell samples. The cells were incubated with the compounds (in triplicate) in a 96-well plate at 37 °C with CO₂ (5%) for two hours. The assay reagent MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (Promega, Madison, WI, USA) was subsequently added and the plate was incubated for four hours. Absorbance readings (at OD₄₉₀) were taken using a kinetic microplate reader (Molecular Devices, Sunnyvale, CA, USA). The quantity of viable cells after treatment with each compound was expressed as a percentage of the viability of DMSO-treated control cells (average of triplicate wells ± standard deviation). The toxicity data was analyzed via a two-way ANOVA, with post hoc Dunnet's multiple comparisons test (P < 0.05), utilizing GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA).

Mohammad H., Kyei-Baffour K., Younis W., Davis D.C., Eldesouky H., Seleem M.N, & Dai M. (2017). Investigation of Aryl Isonitrile Compounds with Potent, Broad-spectrum Antifungal Activity. Bioorganic & medicinal chemistry, 25(11), 2926-2931.

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Cytotoxicity Evaluation of Compounds

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Compounds 1, 5, 22b–d and 25 were assayed at concentrations of 5 μg/mL, 10 μg/mL, 20 μg/mL, and 40 μg/mL against a human embryonic kidney (HEK293) cell line to determine the potential toxic effect to mammalian cells in vitro. Cells were cultured in Dulbeco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO, USA) with 10% fetal bovine serum (USA Scientific, Inc.) at 37 °C with 5% CO₂. Controls received DMSO alone at a concentration equal to that in drug-treated cell samples. The cells were incubated with the compounds in a 96-well plate at 37 °C and 5% CO₂ for 2 hours prior to addition of the assay reagent MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (Promega, Madison, WI, USA). Absorbance readings (at OD₄₉₀) were taken using a kinetic microplate reader (Molecular Devices, Sunnyvale, CA, USA). The quantity of viable cells after treatment with each compound was expressed as a percentage of the viability of DMSO-treated control cells.

Mohammad H., Narasimha Reddy P.V., Monteleone D., Mayhoub A.S., Cushman M, & Seleem M.N. (2015). Synthesis and antibacterial evaluation of a novel series of synthetic phenylthiazole compounds against methicillin-resistant Staphylococcus aureus (MRSA). European journal of medicinal chemistry, 94, 306-316.

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Cytotoxicity Assay of Compounds

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Compounds 1–3 were assayed at concentrations of 5 μg/mL, 10 μg/mL, 20 μg/mL, and 40 μg/mL against a human colorectal (HRT-18) cell line to determine their effects to mammalian cells in vitro, as described elsewhere^{45 (link)}. Cells were cultured in RPMI-1640 medium with 10% fetal horse serum at 37 °C with 5% CO₂. Cells were incubated with compounds in 96-well plates at 37 °C and 5% CO₂ for either 2 or 24 hours prior to addition of the assay reagent MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (Promega, Madison, WI, USA). Absorbance readings (at OD₄₉₀) were taken using a kinetic microplate reader (Molecular Devices, Sunnyvale, CA, USA). The quantity of viable cells after treatment with each compound is expressed as a percentage of the viability of untreated cells.

Mohammad H., Younis W., Chen L., Peters C.E., Pogliano J., Pogliano K., Cooper B., Zhang J., Mayhoub A., Oldfield E., Cushman M, & Seleem M.N. (2017). Phenylthiazole Antibacterial Agents Targeting Cell Wall Synthesis Exhibit Potent Activity In Vitro and In Vivo against Vancomycin-resistant Enterococci. Journal of medicinal chemistry, 60(6), 2425-2438.

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Cytotoxicity-Free Gelatin Formulation

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To select cytotoxicity-free samples for the gelatin formulation, a total of three buffers (6 mM sodium phosphate + 150 mM NaCl + 10% glycerol [pH 7.2]; 20 mM Tris + 25 mM NaCl + 2.5% glycerol [pH 8.0]; and 6 mM sodium phosphate + 150 mM NaCl + 15% sucrose [pH 7.4]) and gelatin samples of various MWs were evaluated. Briefly, each sample was serially diluted twofold in DMEM (Dulbecco’s modified Eagle’s medium) and dispensed at 50 µL/well (triplicate) into a 96-well plate, followed by the addition of 10,000 Vero cells per well and incubation at 37 °C for 72 h. A volume of 20 µL of MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) from Promega (Cat #G3580) was added and incubated at 37 °C for 3 h, followed by absorbance measurement at 490 nm on a plate reader (Ultramark Microplate Reader, Bio-Rad). The survival rate was determined using the formula [Survival rate = (Sample O.D.- Blank)/(Mock OD- Blank) × 100].

Kadji F.M., Kotani K., Tsukamoto H., Hiraoka Y, & Hagiwara K. (2022). Stability of enveloped and nonenveloped viruses in hydrolyzed gelatin liquid formulation. Virology Journal, 19, 94.

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Cell Viability Assay for AML Cells

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AML cell lines or patient-derived cells were treated in a 96-well plate at 0.2-6 × 10⁵ cells per well for 24-72 hours. Patient-derived CD34⁺ primary cells were cultured in 96-well plates coated with collagen in the presence of 10 ng/ml IL-3, 10 ng/ml IL-6, 10 ng/ml SCF and 10 ng/ml GM-CSF. MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (Promega, Madison, WI) was incubated with drug-treated cells according to the manufacturer’s instructions. Plates were read by using a BioTek synergy H4 hybrid multimode microplate reader (Thermo Fisher Scientific) at 490 nM. Combenefit software was used to calculate synergy scores for pairwise combinations of drug doses.

Zhang P., Brinton L., Williams K., Sher S., Orwick S., Tzung-Huei L., Mims A., Coss C., Kulp S., Youssef Y., Chan W.K., Mitchell S., Mustonen A., Cannon M., Phillips H., Lehman A., Kauffman T., Beaver L., Canfield D., Grieselhuber N., Alinari L., Sampath D., Yan P.S., Byrd J.C., Blachly J.S, & Lapalombella R. (2021). Targeting DNA damage repair functions of two histone deacetylases, HDAC8 and SIRT6, sensitizes acute myeloid leukemia to NAMPT inhibition. Clinical cancer research : an official journal of the American Association for Cancer Research.

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