Scribd Logo
Upload

Welcome to Scribd!

  • 132 views

    MTT Assay

    Uploaded by

    Hameedha
    The MTT assay is a colorimetric assay that measures cell viability and proliferation. It works by utilizing the ability of viable cells to reduce yellow tetrazolium MTT into purple formazan crystals through the action of mitochondrial enzymes. The amount of formazan produced is directly proportional to the number of living cells and can be quantified by measuring absorbance. The steps involve adding MTT to cells, incubating to allow formazan formation, solubilizing the crystals, and measuring absorbance to determine cell viability. Parameters like MTT concentration and incubation time must be optimized for each experiment.

    Copyright:

    © All Rights Reserved
    Download as DOC, PDF, TXT or read online from Scribd

    MTT Assay

    Uploaded by

    Hameedha
    132 views2 pages
    The MTT assay is a colorimetric assay that measures cell viability and proliferation. It works by utilizing the ability of viable cells to reduce yellow tetrazolium MTT into purple formazan crystals through the action of mitochondrial enzymes. The amount of formazan produced is directly proportional to the number of living cells and can be quantified by measuring absorbance. The steps involve adding MTT to cells, incubating to allow formazan formation, solubilizing the crystals, and measuring absorbance to determine cell viability. Parameters like MTT concentration and incubation time must be optimized for each experiment.

    Original Description:

    Experimental assay

    Original Title

    MTT ASSAY

    Copyright

    © © All Rights Reserved

    Available Formats

    DOC, PDF, TXT or read online from Scribd

    Did you find this document useful?

    Is this content inappropriate?

    The MTT assay is a colorimetric assay that measures cell viability and proliferation. It works by utilizing the ability of viable cells to reduce yellow tetrazolium MTT into purple formazan crystals through the action of mitochondrial enzymes. The amount of formazan produced is directly proportional to the number of living cells and can be quantified by measuring absorbance. The steps involve adding MTT to cells, incubating to allow formazan formation, solubilizing the crystals, and measuring absorbance to determine cell viability. Parameters like MTT concentration and incubation time must be optimized for each experiment.

    Copyright:

    © All Rights Reserved
    Download as DOC, PDF, TXT or read online from Scribd
    Download as doc, pdf, or txt
    132 views2 pages

    MTT Assay

    Uploaded by

    Hameedha
    The MTT assay is a colorimetric assay that measures cell viability and proliferation. It works by utilizing the ability of viable cells to reduce yellow tetrazolium MTT into purple formazan crystals through the action of mitochondrial enzymes. The amount of formazan produced is directly proportional to the number of living cells and can be quantified by measuring absorbance. The steps involve adding MTT to cells, incubating to allow formazan formation, solubilizing the crystals, and measuring absorbance to determine cell viability. Parameters like MTT concentration and incubation time must be optimized for each experiment.

    Copyright:

    © All Rights Reserved
    Download as DOC, PDF, TXT or read online from Scribd
    Download as doc, pdf, or txt
    of 2

    MTT ASSAY

    Principle:
    The

    MTT

    (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

    bromide)

    tetrazolium

    reduction assay was the first homogeneous cell viability assay developed for a 96-well format
    that was suitable for high throughput screening (HTS). The MTT tetrazolium assay
    technology has been widely adopted and remains popular in academic labs as evidenced by
    thousands of published articles. The MTT substrate is prepared in a physiologically balanced
    solution, added to cells in culture, usually at a final concentration of 0.2 - 0.5mg/ml, and
    incubated for 1 to 4 hours. The quantity of formazan (presumably directly proportional to the
    number of viable cells) is measured by recording changes in absorbance at 570 nm using a
    plate reading spectrophotometer. A reference wavelength of 630 nm is sometimes used, but
    not necessary for most assay conditions.
    Viable cells with active metabolism convert MTT into a purple colored formazan product
    with an absorbance maximum near 570 nm . When cells die, they lose the ability to convert
    MTT into formazan, thus color formation serves as a useful and convenient marker of only
    the viable cells. The exact cellular mechanism of MTT reduction into formazan is not well
    understood, but likely involves reaction with NADH or similar reducing molecules that
    transfer electrons to MTT. Speculation in the early literature involving specific mitochondrial
    enzymes has led to the assumption mentioned in numerous publications that MTT is
    measuring mitochondrial activity.
    The formazan product of the MTT tetrazolium accumulates as an insoluble precipitate inside
    cells as well as being deposited near the cell surface and in the culture medium. The formazan
    must be solubilized prior to recording absorbance readings. A variety of methods have been
    used to solubilize the formazan product, stabilize the color, avoid evaporation, and reduce
    interference by phenol red and other culture medium components. Various solubilization
    methods include using: acidified isopropanol, DMSO, dimethylformamide, SDS, and
    combinations of detergent and organic solvent. Acidification of the solubilizing solution has
    the benefit of changing the color of phenol red to yellow color that may have less interference
    with absorbance readings. The pH of the solubilization solution can be adjusted to provide

    maximum absorbance if sensitivity is an issue; however, other assay technologies offer much
    greater sensitivity than MTT.
    The amount of signal generated is dependent on several parameters including: the
    concentration of MTT, the length of the incubation period, the number of viable cells and
    their and metabolic activity. All of these parameters should be considered when optimizing
    the assay conditions to generate a sufficient amount of product that can be detected above
    background.
    Reagent Preparation
    MTT Solution
    1.Dissolve MTT in Dulbeccos Phosphate Buffered Saline, pH=7.4 (DPBS) to 5 mg/ml.
    2.Filter-sterilize the MTT solution through a 0.2 M filter into a sterile, light protected
    container.
    3.Store the MTT solution, protected from light, at 4C for frequent use or at -20C for long
    term storage.
    Solubilization Solution
    1.Choose appropriate solvent resistant container and work in a ventilated fume hood.
    2.Prepare 40% (vol/vol) dimethylformamide (DMF) in 2% (vol/vol) glacial acetic acid.
    3.Add 16% (wt/vol) sodium dodecyl sulfate (SDS) and dissolve.
    4.Adjust to pH = 4.7
    5.Store at room temperature to avoid precipitation of SDS. If a precipitate forms, warm to
    37C and mix to solubilize SDS.
    MTT Assay Protocol
    1.Prepare cells and test compounds in 96-well plates containing a final volume of
    100 l/well.
    2.Incubate for desired period of exposure. And Add 10 l MTT Solution per well to achieve a
    final concentration of 0.45 mg/ml.
    3.Incubate 1 to 4 hours at 37C.
    4.Add 100 l Solubilization solution to each well to dissolve formazan crystals.
    5.Mix to ensure complete solubilization.
    6.Record absorbance at 570 nm.

    You might also like