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XTT Cell Viability Kit Protocol

A. Reagent Preparation

  1. Thaw reagents just before experiment.

NOTE: Precipitation may occur when reagents are stored at −20°C. Make sure reagents are clear prior to use. If necessary, warm reagents to 37°C to reconstitute.

  1. Add electron coupling solution to XTT Reagent (1:50 volume ratio) to make XTT detection solution. For example, each 96-well plate needs 5 ml XTT solution and 0.1 ml electron coupling solution.

B. XTT Assay

  1. Add 50 µl XTT detection solution to each well of 96-well plate (which contains 100–200 µl/well culture medium) and return plate to incubator.
  2. Read absorbance at 450 nm.

NOTE: The optimal incubation time for this assay depends on experimental setup, such as: cell type, cell number, and treatment. Optimization of incubation time can be determined by reading one plate at various time points after addition of XTT detection solution (featured below).

Figure 1. C2C12 cells were seeded at varying density in a 96-well plate and incubated overnight.

posted October 2012

protocol id: 46

Product Includes Quantity (with Count) Solution Color
XTT Reagent 2 x 25 ml Yellowish
Electron Coupling Solution 2 x 0.5 ml Yellow

Product Description

The XTT Cell Viability Assay Kit is a colorimetric assay that detects the cellular metabolic activities. During the assay, the yellow tetrazolium salt XTT is reduced to a highly colored formazan dye by dehydrogenase enzymes in metabolically active cells. This conversion only occurs in viable cells and thus, the amount of the formazan produced is proportional to viable cells in the sample. The formazan dye formed in the assay is soluble in aqueous solution and can be quantified by measuring the absorbance at wavelength 450 nm using a spectrophotometer. An electron coupling reagent, such as PMS (N-Methylphenazonium methyl sulfate), can significantly improve the efficiency of XTT reduction in cells.


Specificity / Sensitivity

The XTT Cell Viability Kit detects formazan dye produced from XTT conversion by mitochondrial enzymes in cells. Because these mitochondrial enzymes are inactivated shortly after cell death, the orange colored formazan dye only appears in viable cells. This XTT Cell Viability Kit is expected to work in most cells lines. Variable with cell type and incubation time employed in the assay, 0.2-2x104 cells/well should be sufficient for most experiments. For the best result, a cell number titration (as shown in Figure 1) is recommended.


Cell viability and proliferation assays are widely used in drug discovery for the study of growth factors, cytokines, and cytotoxic agents. High throughput screening, in early drug discovery compound screening and in later drug safety and toxicity studies, requires a reliable, sensitive, and simple assay with the ability to analyze a large number of samples. Colorimetric cell viability assays using tetrazolium salt, such as MTT, XTT, WST-1, etc. were developed based on live cells reduction of tetrazolium salt into highly colored formazan compounds (1,2). In contrast to cell proliferation assays, such as radioactive thymidine or BrdU labeling of DNA in live cells followed by quantification of the incorporated thymidine (by quantifying sample radioactivity) or BrdU (using anti-BrdU antibody), the XTT assay doesn’t require radioactive materials, cell fixing, or cell permeabilization. Thus, unlike alternative cellular analysis assays, cells examined in the XTT assay may be used for further analysis.


1.  Scudiero, D.A. et al. (1988) Cancer Res 48, 4827-33.

2.  Roehm, N.W. et al. (1991) J Immunol Methods 142, 257-65.



For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

9095
XTT Cell Viability Kit