195 °C (dec.) (lit.)
2-methoxyethanol: soluble 20 mg/mL
ethanol: soluble 20 mg/mL
H2O: 5 mg/mL
diagnostic assay manufacturing
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If available for a given product, the recommended re-test date or the expiration date can be found on the Certificate of Analysis.
We test the solubility of MTT in water at a concentration of 5 mg/mL. For the MTT viability assay, we dissolve Product No. M5655 at a concentration of 5 mg/ml in RPMI-1640 without phenol red. This medium is available as a powder (Product No.R8755) or liquid (Product No. R7509). MTT can be solubilized in any culture media or balanced salt solution that does not contain phenol red as a pH indicator.
Reconstituted MTT solution is stable for at least 6 months when stored frozen (-20 °C). Storage of reconstituted MTT solution at 2-8°C for more than 2 weeks may cause decomposition and yield erroneous results.
Mitochondrial dehydrogenases of viable cells cleave the tetrazolium ring of the MTT, yielding purple MTT formazan crystals. These crystals are not soluble in aqueous solutions; they may be dissolved in acidified isopropanol (0.1 N HCl in anhydrous isopropanol). If this does not lyse the cells, Triton X-100 can be added to the acidified isopropanol. A typical working concentration is 10% Triton X-100 (i.e., 1 volume of 100% Triton X-100 + 9 volumes of acidified isopropanol).
The reference wavelength (630-690 nm) is used to adjust for the optical variation in the wells of the plate. It also corrects for dust and fingerprints that can be present. The assay is read at 570 nm. The reference wavelength needs to be far enough away from the assay wavelength so as not to affect those values.
It is recommended that flat bottom multi-well plates be used for the MTT assay. The use of tissue culture treated plates is only important if necessary for good cell growth. If cells are grown in larger plates, the supernatant after cell lysis can be transferred to a 96 well plate for reading.
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The MTT asay is a method to assess cell viability. This assay is a semiquantitative assay. The assay is used to compare the viability changes in treated cells to untreated cells. The absorbance is indicative of the cell number. The higher the absorbance, the greater the number of viable cells present. Most researchers compare the absorbance of the two samples as a ratio (ABS treated cells/ABS untreated cells) to get a fold increase/decrease in cell number.
Products M2128 or M5655, Thiazolyl Blue Tetrazolium Bromide can be used for staining dehydrogenase in tissue sections. See the method below. Reagent PreparationMTT (2 mg/ml distilled water) 2.5 ml0.2 M Tris buffer, pH 7.4 2.5 ml0.5 M cobalt chloride 0.5 ml0.05 M magnesium chloride 1.0 mlDistilled water 2.5 mlThe pH of this solution is adjusted to 7.0 if necessary. Aliquot and store at -20°C until required. Sample PreparationCut cytostat sections at 5 to 7 microns. Do not fix. Method:1. Incubate sections in appropriate incubation solution at 37°C for 30-60 minutes.2. Transfer sections to 15% formol saline, 15 minutes.3. Wash in distilled water4. Counterstain in 2% methyl green if required.5. Wash in distilled water.6. Mount in glycerin Jelly. ResultsEnzyme: Black formazan deposits with MTT Source: Bancroft and Gamble, Theory and Practice of Histological Techniques, Fifth Edition. Churchill Livingstone, London, 2002, page 610.
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