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CT01-5 Sigma-AldrichMTT Reagent A

Useful in the study of cell & mitochondrial health, MTT is a pale yellow substrate that is cleaved by living cells to yield a dark blue formazan product.

MTT Reagent A MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.

SDS
CoA
References
Data Sheet

Key Applications: Activity Assay

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Overview

Replacement Information

Key Spec Table

Key Applications
ACT

Description
Catalogue Number	CT01-5
Brand Family	Chemicon®
Trade Name	Chemicon
Description	MTT Reagent A
Overview	Reagent A: MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), 50mg/vial. MTT is a pale yellow substrate that is cleaved by living cells to yield a dark blue formazan product. This process requires active mitochondria, and even freshly dead cells do not cleave significant amounts of MTT. Reagent A is a component of Chemicon's MTT assay kit, Catalog Number CT01/CT02.
Materials Required but Not Delivered	• Solution B (not provided): PBS pH 7.4, 60 mL • Solution C (not provided): Color development solution (isopropanol with 0.04 N HCl), 500 mL

References

Product Information
Quality Level	MQ100

Applications
Application	Useful in the study of cell & mitochondrial health, MTT is a pale yellow substrate that is cleaved by living cells to yield a dark blue formazan product.
Key Applications	Activity Assay
Application Notes	Procedure: 1. Carry out a lymphokine, mitogen, or complement-mediated cytotoxicity assay using standard methods, in 96-well flat-bottomed tissue culture plates of good optical quality (e.g. Falcon). The final volume of tissue culture medium in each well should be 0.1 mL, and the medium (e.g. RPMI or DMEM) may contain up to 10% Fetal Bovine Serum. 2. At the end of the assay add 0.01 mL AB Solution (MTT) to each well. Mix by tapping gently on the side of the tray. 3. Incubate at 37°C for cleavage of MTT to occur. Optimal times may vary according to the assay, but four hours is suitable for most purposes. At the end of this time, the MTT formazan produced in wells containing live cells will appear as black, fuzzy crystals on the bottom of the well. 4. Add 0.1 mL Solution C to each well. Mix thoroughly by repeated pipetting with a multichannel pipettor. The HCl converts the phenol red in tissue culture medium to a yellow color that does not interfere with MTT formazan measurement. The isopropanol dissolves the formazan to give a homogeneous blue solution suitable for absorbance measurement. 5. Within an hour, measure the absorbance on an ELISA plate reader with a test wavelength of 570 nm and a reference wavelength of 630 nm. After several hours at room temperature, serum proteins may begin to precipitate due to the acid/alcohol. Chilling the plates will hasten the precipitation. If the plates must be stored before measuring, keep at 4° C before adding the acid/alcohol, then warm to room temperature and add acid/alcohol just before reading. Results: The MTT assay will normally detect 200 to 50,000 cells of a typical cell line, although 1,000 to 50,000 is the useful range. This number may vary for other cell types. Cytotoxic assays should be set up so that the control, unlysed cells give a signal of 0.2 to 0.4, and proliferation assays should yield a similar value at plateau concentrations. This corresponds to about 20-50,000 cells per well with a typical cell line. Absorbance is directly proportional to the number of cells; actual cells do not absorb significantly, even up to concentrations of l x 10⁶ cells/mL.

Biological Information

Physicochemical Information

Dimensions

Materials Information

Toxicological Information

Safety Information according to GHS

Safety Information

Product Usage Statements
Usage Statement	Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Storage and Shipping Information
Storage Conditions	Maintain at 2-8°C for up to six months. The Reagent A / Solution B mixture is stable at 2-8°C for up to two weeks. Reagent Preparation: For every 1,000 assays to be performed, add 10 mL Solution B to one vial of Reagent A. Mix well, sterile filter and keep in the dark at 4° C until used. Note: It may take overnight to dissolve. Do not heat solution. When absolutely required to dissolve crystals, adjust pH with 1-2 drops of HCl. The AB mixture is stable for several weeks under these conditions.

Storage and Shipping Information

Storage Conditions

Maintain at 2-8°C for up to six months. The Reagent A / Solution B mixture is stable at 2-8°C for up to two weeks.

Reagent Preparation:
For every 1,000 assays to be performed, add 10 mL Solution B to one vial of Reagent A. Mix well, sterile filter and keep in the dark at 4° C until used. Note: It may take overnight to dissolve. Do not heat solution. When absolutely required to dissolve crystals, adjust pH with 1-2 drops of HCl. The AB mixture is stable for several weeks under these conditions.

Packaging Information
Material Size	5 vials

Transport Information

Supplemental Information

Specifications

Global Trade Item Number
Catalogue Number	GTIN
CT01-5	04053252274145

Documentation

MTT Reagent A SDS

Title
Safety Data Sheet (SDS)

MTT Reagent A Certificates of Analysis

Title	Lot Number
REAGENT A FROM COLORIMETRIC (MTT) ASSAY FOR CELL SURVIVAL AND PROLIFERATION - 2120953	2120953
REAGENT A FROM COLORIMETRIC (MTT) ASSAY FOR CELL SURVIVAL AND PROLIFERATION - 2139986	2139986
REAGENT A FROM COLORIMETRIC (MTT) ASSAY FOR CELL SURVIVAL AND PROLIFERATION - 2145123	2145123
REAGENT A FROM COLORIMETRIC (MTT) ASSAY FOR CELL SURVIVAL AND PROLIFERATION - 2445844	2445844
REAGENT A FROM COLORIMETRIC (MTT) ASSAY FOR CELL SURVIVAL AND PROLIFERATION - 2453140	2453140
REAGENT A FROM -2519298	2519298
REAGENT A FROM -2578092	2578092
REAGENT A FROM -2689131	2689131
REAGENT A FROM -2707828	2707828
REAGENT A FROM -2736659	2736659

References

Reference overview	Pub Med ID
Rapid colorimetric assay for cell viability: application to the quantitation of cytotoxic and growth inhibitory lymphokines. Green, L M, et al. J. Immunol. Methods, 70: 257-68 (1984) 1984 Show Abstract A rapid colorimetric microtiter assay has been developed to detect cytotoxic lymphokines produced by human lymphocytes activated with lectins or tumor cells. The viability of lymphotoxin-treated target cells was detected using a tetrazolium dye that is reduced to a blue formazan by living but not dead cells. The amount of dye formed was quantitated using a microplate spectrophotometer (ELISA plate reader) and visual observations confirmed the amount of formazan dye produced was directly proportional to the number of viable target cells. The advantages of using this colorimetric method are that it requires no washing steps or radioisotopes and its precision and rapidity. Optimal conditions were established using the murine L929 and human ESH -5L cell lines as target cells for detecting lymphotoxins produced by human lymphocytes. The data indicate that the L929 cell line was 10-50-fold more sensitive than the ESH -5L line to the lytic activity of cytotoxins produced by human phytohemagglutinin-P-activated T lymphocytes, or the cytotoxins produced by peripheral blood lymphocytes stimulated with various tumor cell lines. This assay system was also useful in detecting antibodies capable of neutralizing lymphotoxin activity and thus should be a suitable method to aid in the molecular characterization of these lymphokines.	6609997

Reference overview

Pub Med ID

Data Sheet

Title
REAGENT A FROM COLORIMETRIC (MTT) ASSAY FOR CELL SURVIVAL AND PROLIFERATION

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