Methyl thiazolyl tetrazolium (mtt)

Manufactured by Agilent Technologies

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The MTT is a laboratory equipment product designed for cell proliferation and cytotoxicity assays. It is a colorimetric assay that measures the activity of cellular enzymes, which can indirectly reflect the number of viable cells in a sample.

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68 protocols using methyl thiazolyl tetrazolium (mtt)

Evaluating Cell Toxicity of Antiviral Compounds

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The cell toxicity of 6MMPr and RIB was tested on growing cells via in situ mitochondrial reduction of a tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, USA). Briefly, VerodogSLAM cells (1 × 10⁴ cells/well) were seeded on 96-well microplates for 24 h. The medium was replaced with 200 μL fresh DMEM containing different concentrations of the compounds. After 72 h of incubation at 37 °C, the culture medium was removed and replenished with 50 μL of MTT working solution (1 mg/mL) to each well and the microplate was incubated for 4 h. MTT formazan crystals were solubilized by adding DMSO (dimethyl sulfoxide) and the optical densities were determined spectrophotometrically with an absorbance microplate reader (BioTek, ELX800, Winooski, Vermont, USA) at 540 nm. Cell viability was calculated by subtracting the optical density fraction of treated cells from the untreated cells. The cytotoxic concentration for 50% of the cell culture (CC₅₀) was expressed as the compound concentration required to reduce the absorbance of treated cells by 50% in comparison to control cells. CC₂₀ was defined as the limit point for treatment with the antiviral molecules [22 (link)]. At least two independent experiments were performed with eight replicates for each concentration level.

de Carvalho O.V., Félix D.M., de Camargo Tozato C., Fietto J.L., de Almeida M.R., Bressan G.C., Pena L.J, & Silva-Júnior A. (2017). 6-methylmercaptopurine riboside, a thiopurine nucleoside with antiviral activity against canine distemper virus in vitro. Virology Journal, 14, 124.

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MCDC Inhibits Cell Viability in Vitro

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Cells were seeded at a density of 3,000 cells per well in 96-well microplate and treated with 2–8 μM 1-(9′-methyl-3′-carbazole)-3, 4-dihydro-β-carboline (MCDC) 24 h after seeding. The testing concentration of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT; BioTek, Winooski, VT, USA) was 0.456 mg/mL. Cells were kept at this concentration of MTT at 37 °C, 5% CO2 for 1.5 h, then dissolved in 100 μL DMSO per well. The absorbance values were measured 48 h after MCDC treatment using a microtiter plate reader (BioTek) at 570 nm.

Ko P.H., Shen Y.C., Murugan K., Huang C.W., Sivakumar G., Pal P., Liao C.C., Luo K.S., Chuang E.Y., Tsai M.H, & Lai L.C. (2019). Macrophage Migration Inhibitory Factor Acts as the Potential Target of a Newly Synthesized Compound, 1-(9′-methyl-3′-carbazole)-3, 4-dihydro-β-carboline. Scientific Reports, 9, 2147.

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Cell Proliferation Assays: MTT and Alamar Blue

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The proliferation rates of each cell were determined by MTT (Sigma, St. Louis, MO, USA) and Alamar Blue (Invitrogen, Carlsbad, CA, USA). For the MTT test, 100 μL of MTT (1 mg/ml) solution was added into 96-well plates and incubated at 37 °C for 2 h. After removing media, 100 μL of isopropanol/1N HCl (v:v = 24:1) was added into each well to dissolve formazan crystals, and the absorbance at 570 nm was measured by a microplate reader (BioTek Epoch, Winooski, VT, USA). For the Alamar Blue assay, 100 μL of Alamar Blue (10% in medium) was added into 96-well plates and incubated at 37 °C for 2 h. Then, the absorbance of Alamar Blue solution was measured by a microplate reader (BioTek Epoch) at 600 nm.

Liu J., Hanavan P.D., Kras K., Ruiz Y.W., Castle E.P., Lake D.F., Chen X., O’Brien D., Luo H., Robertson K.D., Gu H, & Ho T.H. (2018). Loss of SETD2 induces a metabolic switch in renal cell carcinoma cell lines toward enhanced oxidative phosphorylation. Journal of proteome research, 18(1), 331-340.

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Assessing 6MMPr Cytotoxicity in Cells

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The cell toxicity of 6MMPr was tested on growing cells via in situ mitochondrial reduction of a tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, USA). Briefly, 24 h-plated Vero (1x10 4 cells/well) and SH-SY5Y cells (4x10 4 cells/well) in 96-well microplates were treated with various concentrations of the test compound. After 72 h (SH-SY5Y cells) and 120 h (Vero cells) of incubation at 37 °C, culture medium was removed and replenished with 50 μL of MTT solution (1 mg/mL) to each well and the microplate was incubated for 4 h. MTT formazan crystals were solubilized by adding DMSO and the optical densities were determined spectrophotometrically with a 96-well plate reader (BioTek, ELX800, Winooski, USA) at 540 nm. As a control for the cytotoxicity test, ribavirin was used at various concentrations (10 to 100 μM ). Cell viability was calculated by subtracting the optical density fraction of treated cells from the untreated cells. Cytotoxic concentration for 50% of cell culture (CC 50 ) was defined as the concentration of the compound that caused a 50% reduction in absorbance. CC 20 was defined as the limit point for treatment with the antiviral molecules [24] .

de Carvalho O.V., Félix D.M., de Mendonça L.R., de Araújo C.M.C.S., de Oliveira Franca R.F., Cordeiro M.T., Silva Júnior A, & Pena L.J. (2017). The thiopurine nucleoside analogue 6-methylmercaptopurine riboside (6MMPr) effectively blocks Zika virus replication. International journal of antimicrobial agents, 50(6).

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Evaluating Cell Viability with T-5224, RSPO2, and AZD5363

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The FPA cells (GT1-1, GH3) were plated into 96-well culture plate with a density of 500 cells/well, and were treated with different doses of T-5224, RSPO2, and AZD5363, respectively, as well as STO-609 (CaMKK inhibitor) and Genipin (aglycone derived from the iridoid glycoside), both of which protect against several types of tumors, including brain tumors. We used MTT (Sigma, St. Louis, Missouri, USA) dissolved in PBS (5 mg/ml) to measure the viability of cells. On the day of measurement, the medium was replaced on fresh DMEM supplemented with 10% FBS and diluted MTT (1:10, 10% MTT), and incubated for 3.5 h at 37 °C. Then, the incubation medium was removed and formazan crystals were dissolved in 200 μl solution of DMSO. We used an ELx800 absorbance microplate reader (BioTek Instruments, VT, USA) to quantify the MTT reduction by measuring light absorbance at 570 nm. Each test was repeated four times.

Zhong S., Wu B., Li J., Wang X., Jiang S., Hu F., Dou G., Zhang Y., Sheng C., Zhao G., Li Y, & Chen Y. (2019). T5224, RSPO2 and AZD5363 are novel drugs against functional pituitary adenoma. Aging (Albany NY), 11(20), 9043-9059.

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Evaluating Metabolic Activity in Porous Scaffolds

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To evaluate the metabolically active MC3T3-E1 cells on and within the porous scaffolds, a MTT assay was conducted to determine cell proliferation. At designated time of 1, 3 and 7 days, the cell-seeded scaffolds and control wells were placed in 400 μl serum-free medium containing 100 μg of the tetrazolium salt MTT (Sigma, USA; 5mg/ml) for the last 4 h of incubation to permit visualization of metabolically active cells. After the incubation, the scaffolds were briefly rinsed in warm PBS and blotted dry. The insoluble purple formazan, a product of mitochondrial MTT metabolism, was extracted from the scaffolds with 1.0 ml ethanol and measured spectrophotometrically for optical density (OD) values at 490 nm in microplate reader (Bio-Tek, USA).

Jia W., Lau G.Y., Huang W., Zhang C., Tomsia A.P, & Fu Q. (2018). Cellular Response to 3-D Printed Bioactive Silicate and Borosilicate Glass Scaffolds. Journal of biomedical materials research. Part B, Applied biomaterials, 107(3), 818-824.

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MTT Cell Proliferation Assay Protocol

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The cell proliferation assay was performed using MTT (Sigma-Aldrich Co., St Louis, MO, USA). Cells were seeded in 96-well plates at various densities (3 × 10⁴/mL, 5 × 10³/mL, and 1.5 × 10³/mL), and treated with different compounds at various concentrations, for different intervals (24, 48, and 72 hours, respectively). The MTT solution was added to each well at the final concentration of 0.5 mg/mL. After incubation for 4 hours at 37°C, the absorbance was measured at 570 nm using a microplate reader (Biotek Instruments, Inc., Winooski, VT, USA). The IC₅₀ values were calculated using Graphpad Prism 5 Software (GraphPad Software, San Diego, CA, USA).

Ji M., Wang L., Chen J., Xue N., Wang C., Lai F., Wang R., Yu S., Jin J, & Chen X. (2018). CAT3, a prodrug of 13a(S)-3-hydroxyl-6,7-dimethoxyphenanthro[9,10-b]-indolizidine, circumvents temozolomide-resistant glioblastoma via the Hedgehog signaling pathway, independently of O6-methylguanine DNA methyltransferase expression. OncoTargets and therapy, 11, 3671-3684.

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Cytotoxicity Evaluation of Bioresorbable Scaffolds

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Human umbilical vein endothelial cells (HUVECs; ATCC, Manassas, VA) were expanded in growth media consisting of Endothelial Cell Growth Kit-VEGF (ATCC^® PCS-100-041) in Vascular Cell Basal Medium (ATCC^® PCS-100-030) under the standard culture condition (37 °C with 5% CO2 in a humid environment) to 80% confluency before passaging. HUVECs at passages 5-7 were used.
The effect of the iodixanol introduction on the cytotoxicity of BRS was evaluated by means of an indirect cytotoxicity assay following ISO 10993-5: 2009. Specifically, the BRS were first sterilized by Ethylene Oxide and 0.5 mL of growth media was incubated with BRS for 24 hours at 37 °C with agitation. Next, the liquid extracts of different BRS were collected and added to HUVECs at sub-confluence in 24-well pates. After incubation for 24 hours, cells were incubated with growth media containing 0.5 mg/mL MTT (Thermo Fisher Scientific, San Jose, CA) for 3 hours. After replacing the MTT solution with DMSO, the plate was shaken for 15 min to solubilize the formazan crystals, and the absorbance of each well was measured at 570 nm using a microplate reader Cytation 5 (BioTek Instruments). The number of viable cells per well was calculated against a standard curve prepared by plating various concentrations of isolated cells, as determined by hemocytometer, in triplicate in the culture plates.

Ding Y., Fu R., Collins C.P., Sun C, & Ameer G.A. (2022). 3D-Printed Radiopaque Bioresorbable Stents to Improve Device Visualization. Advanced healthcare materials, 11(23), e2201955.

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Astrocyte Viability Assay with Histamine

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The astrocytes at 3 × 10⁵/ml were sub-cultured in 10⁻⁷ mol/L histamine for 30 min on 96-well culture plates, which were previously coated by poly-L-lysine or laminin38 (link). After repeated wash with PBS, the cells were incubated with MTT (Sigma, USA, 0.5 mg/mL as final concentration) for 4 h at 37 °C. Then, the supernatant layer was removed, and 100 μL of dimethyl sulfoxide was added to each well. MTT metabolism was quantified spectrophotometrically at 570 nm by a Biotek microplate reader (USA).

Liao R.J., Jiang L., Wang R.R., Zhao H.W., Chen Y., Li Y., Wang L., Jie L.Y., Zhou Y.D., Zhang X.N., Chen Z, & Hu W.W. (2015). Histidine provides long-term neuroprotection after cerebral ischemia through promoting astrocyte migration. Scientific Reports, 5, 15356.

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Cell Viability Assay via MTT

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The in vitro cell viability test was performed with a MTT assay as described previously [43 (link), 44 (link)]. 24 h post transfection, cells were seeded in 96-well culture plates (4 × 10³/well) for 12 h, 24 h, 36 h, 48 h, and 60 h (in triplicate for each condition). 20 µl MTT (Sigma, Saint Louis, USA) stock solution (5 mg/ml) was added to each well and incubated for 2–3 h. Then, the supernatant was removed, followed by the solubilisation of MTT crystals using DMSO and the absorbance was measured at 570 nm using plate reader BioTek Eon (BioTek, Winooski, USA).

Ashok C., Ahuja N., Natua S., Mishra J., Samaiya A, & Shukla S. (2021). E2F1 and epigenetic modifiers orchestrate breast cancer progression by regulating oxygen-dependent ESRP1 expression. Oncogenesis, 10(8), 58.

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