Fluorescent Dye; NAD (P) H-oxidoreductases

1. Preparation of MTT working solution:
MTT was dissolved with PBS to obtain 5 mg/mL of MTT.
2. Cell proliferation test (96-well plate).
2.1 Inoculated cells: The prepared single cells were prepared with a suspension containing 10%FBS and cultured a 96-well plate with 1000-10000 cells per well and a volume of 100μL per well.
2.2 Cultured cells: 37℃. 5% CO2, incubated for 24-72 h.
2.3 Add 10 μL MTT to each well, incubate for 4 h, discard the supernatant (centrifuge first if using suspended cells).
2.4 Add 100 μL DMSO and shake for 10 minutes to completely dissolve the crystal.
2.5 Monitor the increase in absorbance at OD=562 nm with an absorbance board reader.

Tetrazolium salts have become some of the most widely used tools in cell biology for measuring the metabolic activity of cells ranging from mammalian to microbial origin. With mammalian cells, fractionation studies indicate that the reduced pyridine nucleotide cofactor, NADH, is responsible for most MTT reduction and this is supported by studies with whole cells. MTT reduction is associated not only with mitochondria, but also with the cytoplasm and with non-mitochondrial membranes including the endosome/lysosome compartment and the plasma membrane. The net positive charge on tetrazolium salts like MTT and NBT appears to be the predominant factor involved in their cellular uptake via the plasma membrane potential. However, second generation tetrazolium dyes that form water-soluble formazans and require an intermediate electron acceptor for reduction (XTT, WST-1 and to some extent, MTS), are characterised by a net negative charge and are therefore largely cell-impermeable. Considerable evidence indicates that their reduction occurs at the cell surface, or at the level of the plasma membrane via trans-plasma membrane electron transport. The implications of these new findings are discussed in terms of the use of tetrazolium dyes as indicators of cell metabolism and their applications in cell biology.[1]

The MTT assay is simple, accurate and yields reproducible results. This method has been developed originally by Mossman in 1983. The key component is (3-[4,5-dimethylthiazol-2-yl]- 2,5-diphenyl tetrazolium bromide) or MTT. Mitochondrial dehydrogenases of viable cells cleave the tetrazolium ring, leading to the formation of purple crystals which are insoluble in aqueous solutions. The crystals are re-dissolved in acidified isopropanol and the resulting purple solution is measured spectrophotometrically. An increase or decrease in cell number results in a concomitant change in the amount of formazan formed, indicating the degree of cytotoxicity caused by the test material (IC50).

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A tetrazolium salt has been used to develop a quantitative colorimetric assay for mammalian cell survival and proliferation. The assay detects living, but not dead cells and the signal generated is dependent on the degree of activation of the cells. This method can therefore be used to measure cytotoxicity, proliferation or activation. The results can be read on a multiwell scanning spectrophotometer (ELISA reader) and show a high degree of precision. PMID:6606682

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/The authors/ compared the MTT assay to the standard clonogenic assay and had good agreement of surviving fraction /of murine solid tumor cells/ after radiation doses of 2 and 4 Gy. It is possible, therefore, to adapt the MTT assay for use with cell suspensions prepared directly from fresh murine tumors. This may provide a methodology for the determination of the clinical radiosensitivity of tumors including fresh clinical tumor specimens. PMID:3417490

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A microcytotoxicity assay employing a tetrazolium salt has been adapted for testing the response of human leukemic blast cells to a variety of chemotherapeutic agents. After exposure to various concentrations of drugs, the viability of fresh leukemic blast cells was measured using a tetrazolium salt, MTT, which is converted to blue formazan crystals by living cells. The amount of formazan produced was quantitated using a microtiter plate spectrophotometer. https://pubchem.ncbi.nlm.nih.gov/compound/64965

Toxicological Information

Interactions
KB cells were treated with MTT at final concentrations up to 0.2 mg/ml alone or in combination with methotrexate (10 and 32 uM). When used alone, as little as 0.025 mg/ml increased population doubling time from 26 hr (control) to 96 hr. At 0.2 mg/ml or higher, total cell number declined. MTT, 0.1 mg/ml, reduced the number of cells in S and G2/M phases after 8 hr treatment. However, no significant effect was seen at 2 and 4 hr. The effect of 0.2 mg/ml MTT was more immediate and more pronounced, resulting in accumulation of cells in G0/G1 and S phases. MTT in combination with methotrexate produced a more significant perturbation in the KB cell cycle.

...Phenazine methosulfate (PMS) and MTT are often used in combination: however, PMS did not potentiate the mutagenicity of MTT. Venitt S, Crofton-Sleigh; Mutation Research/Genetic Toxicology 68 (2): 107-116 (1979)

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Antidote and Emergency Treatment
Basic treatment: Establish a patent airway. Suction if necessary. Watch for signs of respiratory insufficiency and assist ventilations if needed. Administer oxygen by nonrebreather mask at 10 to 15 L/min. Monitor for pulmonary edema and treat if necessary ... . Monitor for shock and treat if necessary ... . Anticipate seizures and treat if necessary ... . For eye contamination, flush eyes immediately with water. Irrigate each eye continuously with normal saline during transport ... . Do not use emetics. For ingestion, rinse mouth and administer 5 ml/kg up to 200 ml of water for dilution if the patient can swallow, has a strong gag reflex, and does not drool ... . Cover skin burns with dry sterile dressings after decontamination ... . /Poison A and B/

Advanced treatment: Consider orotracheal or nasotracheal intubation for airway control in the patient who is unconscious, has severe pulmonary edema, or is in respiratory arrest. Positive pressure ventilation techniques with a bag valve mask device may be beneficial. Monitor cardiac rhythm and treat arrhythmias as necessary ... . Start an IV with D5W /SRP: "To keep open", minimal flow rate/. Use lactated Ringer's if signs of hypovolemia are present. Watch for signs of fluid overload. Consider drug therapy for pulmonary edema ... . For hypotension with signs of hypovolemia, administer fluid cautiously. Watch for signs of fluid overload ... . Treat seizures with diazepam (Valium) ... . Use proparacaine hydrochloride to assist eye irrigation ... . /Poison A and B/

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Non-Human Toxicity Excerpts
/GENOTOXICITY/ Tetrazolium salts, in combination with phenazine methosulfate (PMS) are widely used laboratory reagents. PMS, and 3 tetrazolium salts (MTT, [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide]; TTC,[2,3,5-triphenyl tetrazolium chloride]; and NBT, [2,2'-di-p-nitrophenyl-5,5'-diphenyl-3,3'-(3,3'-dimethoxy-4,4' were tested for mutagenicity in a range of Escherichia coli WP2 (trp-) and Salmonella typhimurium ... . Without S-9, PMS was mutagenic to E. coli uvrApKM101 /and/ S. typhimurium TA 100 and TA 98 giving (in the dose range 0.5-10 ug/plate) linear dose response curves with slopes of 2.3, 1.3 and 0.5 revertants/nmol respectively. Addition of /rat liver/ S-9 drastically reduced the mutagenicity of PMS in these bacteria. MTT, in the absence of S-9, was mutagenic to E. coli uvrApKM101, S.typhimurium TA 100 and TA 98, giving linear dose-response curves with slopes of 3.8, 12.1 and 1.0 respectively, at doses ranging from 0.5 to 10 or 50 ug/plate. Addition of S-9 markedly reduced the mutagenicity of MTT. MTT was very weakly mutagenic in E. coli WP2 (0.22 revertants/nmol), significantly more mutagenic in WP2 uvrA (1.29 revertants/nmol), and non-mutagenic in WP2 lexA (negative slope due to toxicity), suggesting that MTT causes excisable misrepair DNA damage. PMS and MTT are often used in combination: however, PMS did not potentiate the mutagenicity of MTT.

/ALTERNATIVE IN VITRO TESTS/ ... /The authors/... investigated the effects of MTT on cell growth and cell cycle distribution using DNA flow cytometry. KB cells were treated with MTT at final concentrations up to 0.2 mg/ml alone or in combination with methotrexate (10 and 32 uM). When used alone, as little as 0.025 mg/ml increased population doubling time from 26 hr (control) to 96 hr. At 0.2 mg/ml or higher, total cell number declined. MTT, 0.1 mg/ml, reduced the number of cells in S and G2/M phases after 8 hr treatment. However, no significant effect was seen at 2 and 4 hr. The effect of 0.2 mg/ml MTT was more immediate and more pronounced, resulting in accumulation of cells in G0/G1 and S phases. MTT in combination with methotrexate produced a more significant perturbation in the KB cell cycle. Although the MTT assay measures cell viability and proliferation via mitochondrial dehydrogenases, it showed definite effects on cellular DNA content and cell proliferation. These observations question the credibility of such an assay, indicating it may be measuring combined effects of MTT and drug and not drug alone.

/OTHER TOXICITY INFORMATION/ Despite widespread use of various tetrazolium assays, the mechanisms of bioreduction of these compounds have not been fully elucidated. /The authors/ investigated the capacity of tetrazolium salts to penetrate through intact cell plasma membranes. 5-Cyano-2,3-ditolyl tetrazolium chloride (CTC) and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) tetrazolium salts appear to represent examples of species that are reduced by different mechanisms. /The authors/ provide evidence suggesting that MTT readily crosses intact plasma membranes and is reduced intracellularly. MTT appears to be reduced by both plasma membrane and intracellular reductases; reducing cells are not damaged and remain metabolically active for at least 45 min. In contrast, CTC remains extracellular with respect to viable cells and thus requires plasma membrane permeable electron carrier to be reduced efficiently. However, reduction of CTC in the presence of an electron carrier inflicts damage on plasma membranes. The intracellular vs extracellular sites of reduction of tetrazolium salts were established on the basis of deposition of formazans. Crystals of formazan were detected using fluorescence or backscattered light confocal laser microscopy. /The authors/ postulate that the capacity of a tetrazolium salt to cross intact plasma membranes constitutes an important experimental variable which needs to be controlled in order to correctly interpret the outcome of tetrazolium assays designed to measure cellular production of oxygen radicals, activity of mitochondrial, cytosolic, or outer membrane reductases, etc. PMID:10900139

[1]. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol Annu Rev. 2005;11:127-52.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide is the bromide salt of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium. It has a role as a dye and a colorimetric reagent. It contains a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium.

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Sources/Uses
Used in biological assays;


Tetrazolium salts are used for the detection of dehydrogenase activity or any other enzyme systems where redox equivalents are generated. Due to this feature, they are an extremely useful tool in academic and clinical research as well as for many diagnostic applications, eg: (1) aids and cancer research, cell and molecular biology: for testing cell proliferation and cell viability and for cytotoxicity tests (2) Enzyme diagnostics and clinical chemistry: for the assay of dehydrogenase activity and other enzyme systems which form redox equivalents; for the determination of dehydrogenase substrate concentrations; for the detection of glucose, ethanol, glycerol and other substrates which develop redox equivalents in a coupled reaction (3) Immuno histochemistry: for the detection of alkaline phosphatase (4) Histology and pathology: for the histochemical detection of dehydrogenase (5) In seed nurseries: for testing seed viability. SERVA Tetrazolium Salts - Data Sheet. Available from, as of March 28, 2005: https://www.serva.de/products/latest/tetrazolium.shtml

Tetrazolium salts are widely used for detecting redox potential of cells for viability, cytotoxicity and proliferation assays. ... Reduction of MTT remains the most common assay for tetrazolium salt-based viability testing.

Tetrazolium salts for detecting redox potential in living cells and tissues: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Applications: superoxide generation by fumarate reductase and nitric oxide synthase; mitochondrial dehydrogenase activity; cell viability and proliferation; neuronal cell death; platelet activation; tumor cell adhesion and invasion; multidrug resistance; in vitro toxicity testing /from table/

The MTT assay is frequently used in anti-HIV and antineoplastic drug screening for the evaluation of cell survival and proliferation.

C18H16BRN5S

414.32

413.03

C, 52.18; H, 3.89; Br, 19.29; N, 16.90; S, 7.74

298-93-1

13146-93-5 (parent)

64965

Light yellow to yellow solid powder

195 °C (dec.)(lit.)

0.288

0

5

3

25

410

0

AZKSAVLVSZKNRD-UHFFFAOYSA-M

InChI=1S/C18H16N5S.BrH/c1-13-14(2)24-18(19-13)23-21-17(15-9-5-3-6-10-15)20-22(23)16-11-7-4-8-12-16;/h3-12H,1-2H3;1H/q+1;/p-1

2-(3,5-diphenyltetrazol-2-ium-2-yl)-4,5-dimethyl-1,3-thiazole;bromide

Thiazolyl Blue; Thiazolyl Blue Tetrazolium Bromide; 298-93-1; MTT; MMT Tetrazolium; Methylthiazoletetrazolium; Thiazolyl Blue Monotetrazolium; 2348-71-2; Methylthiazolyldiphenyl-tetrazolium bromide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~25 mg/mL (~60.34 mM)
H2O : ~0.88 mg/mL (~2.12 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.02 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.02 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: 6.67 mg/mL (16.10 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)