Performing an MTT assay is easy enough, but there can be pitfalls if one is unfamiliar with the protocol. Here is a brief description of the steps. The MTT tetrazolium assay technology has been widely adopted and remains .. Multiplexing may require a sequential protocol to avoid color. Principle: Rapid colorimetric assay based on the cleavage of the tetrazolium ring of. MTT (3-(4,5-dimethylthazolkyl)-2,5-diphenyl tetrazolium bromide) by.


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Ensure that the same volume of existing media is present for each sample.

MTT assay protocol

Wrap plate in foil and mtt assay principle on an orbital shaker for 15 minutes. Occasionally, pipetting of the liquid may be required to fully dissolve the MTT formazan.


Read plate within 1 hour. Notes Serum or phenol red present in the culture medium can generate background. If your sample contains serum or phenol red, set up sample background controls: Prepare parallel well s as solvent control and use same volume of solvent as for the treated cells.

Data analysis Average mtt assay principle duplicate reading for each sample.

MTT assay protocol | Abcam

Resazurin can be dissolved in physiological buffers resulting in a deep blue colored solution and added directly to cells in culture in a mtt assay principle format. Viable cells with active metabolism can reduce resazurin into the resorufin product which is pink and fluorescent Figure 7.

Structure of resazurin substrate and the pink fluorescent resorufin product resulting from reduction in viable cells. Addition of an intermediate electron acceptor is not required for cellular resazurin reduction to occur, but it may accelerate signal mtt assay principle.

Resorufin also can be quantified by measuring a change in absorbance; however, absorbance detection is not often used because it is far less sensitive than measuring fluorescence. The resazurin reduction assay is slightly more sensitive than tetrazolium reduction assays and there are numerous reports using the resazurin reduction assay in a miniaturized format for HTS applications The incubation period required to generate an adequate fluorescent signal above background is usually 1to 4 hours and is dependent on the metabolic activity of the particular cell type, the cell density per well, and other assay conditions mtt assay principle the type of culture medium.

The incubation period should be optimized and kept short enough to avoid reagent toxicity but long enough to provide adequate sensitivity. mtt assay principle

  • MTT assay - Wikipedia
  • Cell Viability Assays - Assay Guidance Manual - NCBI Bookshelf

The major advantages of the resazurin reduction assay are that it is relatively inexpensive, it uses a homogeneous format, and it is more sensitive that tetrazolium assays. In addition, resazurin assays can be multiplexed with other mtt assay principle such as measuring caspase activity to gather more information about the mechanism leading to cytotoxicity 28 and Figure 8.

Panel A shows the steps of the sequential multiplex of a resazurin assay to measure viable cell number and a fluorometric caspase 3-assay to detect a marker of apoptosis. Panel B shows the results of treating PC3 human prostate cells with a range of more Multiplexing may require a sequential protocol to avoid color quenching by resazurin or direct chemical interference.

mtt assay principle

For the multiplex example shown in Figure 8mtt assay principle fluorescence must be recorded first, followed by addition of the caspase reagent which contains detergent to lyse cells and reducing compounds to convert remaining resazurin and reduce interference mtt assay principle collecting the second fluorescent signal.

The disadvantages of the resazurin include the possibility of fluorescent interference from compounds being tested and the often overlooked direct toxic effects on the cells Figure 9.

Change in NIH3T3 cell morphology after exposure to resazurin.


Panel A shows a field of cells photographed immediately after addition of the resazurin solution. Panel Mtt assay principle shows the same field of cells photographed after 4 hours of exposure to resazurin.

Some protocols describe exposing cells to resazurin for several hours or even days; however, in some systems, changes in cell morphology can mtt assay principle observed after only a few hours of exposure suggesting interference with normal cell function It is possible that exposure of cells to resazurin depletes reduced forms of nucleotides resulting in cytotoxic effects.