MTT assay absorbance is correlated with the number of cells

XTT and MTT assays for cell viability are simple and optimized colorimetric-based assays that function to evaluate cell proliferation and viability on microplate readers. These assays use the cellular redox potential in metabolically active cells to convert a tetrazolium-based salt reagent—MTT or XTT—to a brightly colored formazan product.

Selection guide for MTT and XTT Assays

 CyQUANT MTT Cell Viability AssayCyQUANT XTT Cell Viability Assay
Assay typeColorimetric mix-and-read assays using the cellular redox potential in metabolically active cells
Use
  • End-point assay for quantitatively determining viability and proliferation
  • Quick, continuous assay for quantitatively determining viability and proliferation
  • Detection of small cell populations
  • Detection of poorly metabolizing cells
Components
  • MTT Reagent (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
  • Sodium Dodecyl Sulfate (SDS)
  • XTT Reagent (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide)
  • Electron Coupling Reagent
Mechanism of detection

Conversion of water-soluble MTT to an insoluble purple-colored formazan


MTT assay mechanism of action

Conversion of water-soluble XTT to a water-soluble orange-colored formazan

XTT assay mechanism of action
SolubilityInsoluble formazan; requires additional solubilization steps with SDS or DMSOSoluble formazan; does not require additional solubilization steps
Sensitivity
Incubation time8+ hours4 hours
Continuous AssayNoYes
Scalability96-well plate96- and 384-well plate
ReproducibilityModerateHigh
Absorbance (nm)570 nm450 nm and 660 nm
Cat. No.V13154X12223

The following stand-alone product is also available: MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Cat. No. M6494).

Protocol for MTT and XTT Assays

CyQUANT MTT cell viability assay

MTT as a measure of cellular redox potential

An MTT assay is a simple method for quantitatively determining cell number using standard absorbance microplate readers. It is a well-established assay that measures for cellular viability and proliferation.

The CyQUANT MTT cell viability assay uses the cellular redox potential of metabolically active cells to reduce water-soluble MTT to an insoluble purple formazan. The formazan is then solubilized with sodium dodecyl sulfate (SDS, included in the assay) and measured on a microplate reader at 570 nm (Figure 1). The concentrated formazan is directly correlated with the number of metabolically active cells (Figure 2).

Figure 1. CyQUANT MTT Cell Viability Assay mechanism.
 

MTT assay absorbance is correlated with the number of cells

Figure 2. Quantitation of Jurkat cells using the CyQUANT MTT Cell Viability Assay Kit. Cells in the parent culture were counted, diluted to the indicated densities in 100 µL volumes, then transferred to wells of a microplate, and incubated for 4 hours with CyQUANT MTT Cell Viability Assay to allow time for adsorption. Absorbance was measured at 570 nm using a microplate reader. Each data point represents the mean value of samples in triplicate. The inset shows the data plotted for the lower cell numbers.

How do I use the MTT cell viability assay?

To use the MTT assay, prepare the MTT stock solution and the SDS-HCl solution, add the MTT solution, incubate for 4 hours at 37ºC, add the SDS-HCl solution, incubate for an additional 4–18 hours in a humidified chamber, then read the absorbance at 570 nm. The use of DMSO as a solubilizing agent may be substituted for the SDS-HCl solution with incubation reduced to 10 minutes at 37ºC and absorbance read at 540 nm (not 570 nm).

See also: MTT Cell Viability Assay Kit Protocol

CyQUANT XTT cell viability assay

XTT as a measure of the cellular redox potential

CyQUANT XTT cell viability assay is a continuous assay for quantitatively determining cell viability and proliferation. XTT has shown to be particularly useful for high throughput screening of antiviral and antitumor agents, and for assessing the effects of cytokines on cell proliferation.

The XTT assay is an easy-to-use, optimized assay that contains the XTT reagent and an Electron Coupling Reagent. The XTT assay uses the cellular redox potential of metabolically active cells to reduce water-soluble XTT to a water-soluble orange-colored formazan. The Electron Coupling Reagent, an electron mediator, captures readily available electrons on the plasma membrane to form a reactive intermediate that aids in the reduction of XTT (Figure 3).

Figure 3. CyQUANT XTT Cell Viability Assay mechanism.
 

The water-soluble formazan product allows for easier quantification of cellular activity and helps reduce overall assay time with a recommended 4-hour incubation for optimal results; however, the assay can be completed in as little as 2 hours without affecting sensitivity. The Electron Coupling Reagent increases sensitivity and improves consistency, displaying an up to 8-fold increase in signal-to-background ratios (Figure 4). Further, the CyQUANT XTT assay does not require cell lysis and uses noninvasive probes allowing for the continuous analysis of cell health with additional functional reagents.

See BioProbes article: An Absorbance-Based Assay for Cell Health and Proliferation
See poster: New and improved cellular health evaluation of 2D and 3D cellular models using microplate reader assays

Figure 4. Viability determination using multiple methods. A549 cells were plated at various concentrations (x-axis). After an overnight incubation, the manufacturer’s instructions were followed for either the CyQUANT XTT Cell Viability Assay, the Cell Proliferation Kit II (Sigma), or MTT Reagent. For the XTT-based assays, the absorbance was read at 450 and 660 nm using the Varioskan LUX instrument. Cell Viability using MTT Reagent required a 540 nm absorbance measurement. The specific absorbance signals were determined and signal-to-background (wells with no cells) ratios were generated.

How do I use the XTT cell viability assay?

Using the CyQUANT XTT cell viability assay is simple. Thaw the XTT Reagent and Electron Coupling Reagent, mix to prepare the working solution, add the solution to your cells, incubate for 4 hours at 37ºC, and read the absorbance.

It is important to use the XTT/Electron Coupling Reagent immediately after preparation as performance and sensitivity will be reduced if the solution is kept at room temperature for an extended period of time or subject to freeze/thaw cycles. To ensure assay performance, the assay consists of separate XTT and Electron Coupling Reagents that should be kept at –20ºC until use.

The XTT-specific absorbance is measured at 450 nm. The 660 nm absorbance reading is used to eliminate the background signal contributed by cell debris or other non-specific absorbance.

Generating drug-dose response curves with CyQUANT XTT and MTT assays

CyQUANT XTT and MTT assays were used to generate drug-dose response curves in cells exposed to gambogic acid, a xanthanoid compound known for its anticancer effects, including inhibiting cell growth by inducing apoptosis and autophagy (1). Comparative drug dose response curves showed IC50 values were similar between the XTT and MTT assay (Figure 5).

Drug-dose curves for treated cells have similar IC50 between the XTT and MTT assay

Figure 5. Comparison of CyQUANT XTT and MTT assay sensitivity. The CyQUANT XTT Cell Viability Assay and a commercially available MTT assay were used according to their respective manufacturers’ protocols to generate drug-dose response curves for A549 cells exposed to increasing concentrations of gambogic acid for ~18 hours at 37°C and 5% CO2. The IC50 value for gambogic acid generated with the CyQUANT XTT assay (3.28 μm) was similar to that obtained with the MTT assay (2.76 μm). Absorbance of the XTT assay product is reported as the absorbance of the sample at 450 nm minus the absorbance of the sample at 660 nm minus the absorbance of the blank at 450 nm. Absorbance of the MTT assay product is reported as the absorbance of the sample at 540 nm minus the absorbance of the blank at 540 nm. All measurements were made using a Thermo Scientific Varioskan LUX Multimode Microplate Reader, and data were analyzed using GraphPad Prism software.
 

Expertly detect fluorescence with Thermo Scientific plate readers

Thermo Scientific plate readers

High-sensitivity fluorescence detection for 96-1,536 samples can be quickly performed on Fluoroskan or Fluoroskan FL Microplate Fluorometer or Varioskan LUX Multimode Microplate Reader using Invitrogen reagents for optimal detection. Take advantage of automatic dynamic range selection to get optimal gain settings for each individual well and automation capabilities for even higher throughput.

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Citations

Resources

Cell Analysis Learning Center—Find educational resources such as application notes, webinars, videos, articles, and more that cover the use of many of our reagents and kits for cell analysis.

Fluorescence SpectraViewer—Online tool for visualization of the excitation and emission of fluorescent reagents; allows for checking spectral compatibility for multiple fluorophores.

BioProbes article

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