CyQUANT XTT and MTT Assays for Cell Viability

	CyQUANT MTT Cell Viability Assay	CyQUANT XTT Cell Viability Assay
Assay type	Colorimetric 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 See example of detection mechanism × CyQUANT MTT Cell Viability Assay Close	Conversion of water-soluble XTT to a water-soluble orange-colored formazan See example of detection mechanism × CyQUANT XTT Cell Viability Assay Close
Solubility	Insoluble formazan; requires additional solubilization steps with SDS or DMSO	Soluble formazan; does not require additional solubilization steps
Sensitivity
Incubation time	8+ hours	4 hours
Continuous assay	No	Yes
Scalability	96-well plate	96- and 384-well plate
Reproducibility	Moderate	High
Absorbance (nm)	570 nm	450 nm and 660 nm
Cat. No.	V13154	X12223

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

See also: MTT Cell Viability Assay Kit Protocol

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.
 

To use the MTT assay, prepare the MTT stock solution and the SDS-HCl solution, add the MTT solution, incubate for four 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 ten minutes at 37ºC and absorbance read at 540 nm (not 570 nm).

See also: MTT Cell Viability Assay Kit Protocol

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CyQUANT XTT cell viability assay is a continuous assay for quantitatively determining cell viability and proliferation using the cellular redox potential in live cells. 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 four-hour incubation for optimal results; however, the assay can be completed in as little as two hours without affecting sensitivity. The Electron Coupling Reagent increases sensitivity and improves consistency, displaying an up to an eight-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.

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 four 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.

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).

1. Liu Y, Chen Y, Lin L, and Li H (2020) Int J Nanomedicine. 15:10385-10399. PMID: 33376327