Catalog No. 30-1011K

Introduction
Background
XTT Assay Kit Results
Comparison of the XTT and MTT Cell Proliferation Assays
Frequently Asked Questions
Ordering
Product Instruction Manual

Tetrazolium salts have been widely used as detection reagents for many years in histochemical localization studies and cell biology assays (1, 2). The second generation tetrazolium dye, XTT (sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt), can be effectively used in cell-based assays to measure cell growth, cytotoxicity, and apoptosis assays (2, 3, 4). XTT is reduced to a soluble, brightly colored orange derivative by a mix of cellular effectors. The sensitivity of an XTT assay is greatly improved by the usage of an intermediate electron carrier, PMS (N-methyl dibenzopyrazine methyl sulfate). PMS helps drive XTT reduction and the formation of its formazan derivative.

• Homogenous Assay. Easy to use with no solubilization step.
• High-Throughput. Fast results, automation-friendly.
• High Sensitivity. Real-time assay results using low cell numbers.
• Reproducible Accuracy. Dye absorbance proportional to cell number.
• Non-Radioactive. Safe, no hazardous waste.
• Convenient storage. The kit is stable for 18 months when stored under refrigeration in the dark.

Background

The XTT cell proliferation assay was first described in 1988 by Scudiero et al. (3) as an effective method to measure cell growth and drug sensitivity in tumor cell lines. XTT is a colorless or slightly yellow compound that when reduced becomes brightly orange (Figure 1). This color change is accomplished by breaking apart the positively-charged quaternary tetrazole ring (2). The formazan product of XTT reduction is soluble and can be used in real-time assays.

XTT is thought to be excluded from entering cells by its net negative charge (2). Considerable evidence suggests that XTT dye reduction occurs at the cell surface facilitated by trans-plasma membrane electron transport. Mitochondrial oxidoreductases are thought to contribute substantially to the XTT response with their reductants being transferred to the plasma membrane (Figure 2). It has been proposed that XTT assays actually measure the pyridine nucleotide redox status of cells (2, 4).

XTT can be used alone as a detection reaction but the results are not optimal. XTT assay results are greatly improved when an intermediate electron acceptor, such as PMS (N-methyl dibenzopyrazine methyl sulfate), is used with XTT (Figure 2). PMS is the Activation Reagent included in the XTT Cell Proliferation Assay Kit. Findings suggest that PMS mediates XTT reduction by picking up electrons at the cell surface, or at a site in the plasma membrane that is readily accessible, and forms a reactive intermediate that then reduces XTT to its highly pigmented formazan product.

References

1. Altman, FP. Tetrazolium salts and formazans. Prog. Histochem. Cytochem. 9: 1-56, 1976.
2. Berridge MV, Herst PM and  Tan AS. Tetrazolium dyes as tools in cell biology: New insights into their cellular reduction. Biotechnology Annual Review 11:127-152, 2005. 
3. Scudiero DA, et al., Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res. 48: 4827-4833, 1988.
4. Marshall NJ, Goodwin CJ and Holt SJ. A critical assessment of the use of microculture tetrazolium assays to measure cell growth and function. Growth Regulation 5: 69-84, 1999.

XTT Assay Kit Results

Scientists at ATCC performed two assays using the XTT Cell Proliferation Assay Kit with L-929 cells. The studies assessed cellular proliferation and viability of L-929 cells (Figure 3) and TNF-α-mediated cytotoxicity of L-929 cells (Figure 4).

Comparison of the XTT and MTT Cell Proliferation Assay Kits

The performance of the XTT Cell Proliferation Assay Kit was compared to that of the MTT Cell Proliferation Assay. Although both are based on the chemical reduction of a tetrazolium salt by a cascade of intracellular enzymes, the two compounds provide researchers with different options for experimental design. Key differences between the two assays are highlighted in the following table:

The mouse fibroblast cell line,L-929 (ATCC CCL-1), was cultured overnight at 37°C in a 5% CO2 incubator. No additional agents were added during the cell culture. The cell proliferation and viability of L-929 cells were assessed by XTT and MTT assays (3 hr assay incubation time). The XTT assay (measured at 475 nm) exhibited higher absorbance values than the MTT assay (measured at 570nm) but showed a similar loss of linearity when greater than 2.5 x 10⁴ L-929 cells were used in the cell proliferation assays.

Frequently Asked Questions

1. Why did the XTT Reagent change color?
   
   It was colorless to pale yellow, but now it's blue-green. There are two potential causes for this problem. The XTT Reagent might have been contaminated by bacteria or cells, or may have been exposed to light. Regardless, you should not use XTT Reagent that is blue-green rather than colorless to pale yellow. Store the XTT Reagent in the dark at -20°C. Be sure to use aseptic technique when dispensing the XTT Reagent.

    

2. Why are the absorbance readings for all of my blanks (i.e., wells containing only growth medium) too high?
   
   There are two potential causes for this problem. The XTT Reagent might have been contaminated by bacteria or cells, or the complete growth medium contains reducing agents such as ascorbic acid. Two possible sources of contamination include the XTT Reagent itself or the use of non-sterile flat-well microtiter plates or pipette tips. Always dispense the XTT Reagent using aseptic technique and perform the assay in a biological hood using sterile microtiter plates and pipette tips. Discard any XTT Reagent that has been inappropriately stored or handled. If the complete growth medium contains ascorbic acid (as do many specialty media), find an alternative medium if possible, or try incubating the plate during the assay in the dark.

    

3. Why are absorbance readings too high in wells containing cells and Activated-XTT Solution?
   
   There are a number of potential causes for this problem. The XTT Reagent could have been contaminated by bacteria or cells, the number of cells per well are too high, or the assay incubation time might be too long. Two possible sources of contamination include the XTT Reagent itself or the use of non-sterile flat-well microtiter plates or pipette tips. Always dispense the XTT Reagent using aseptic technique and perform the assay in a biological hood using sterile microtiter plates and pipette tips. Discard any XTT Reagent that has been inappropriately stored or handled. If contamination can be ruled out, reduce the number of cells per well or incubate the cells for a shorter period of time. If you are performing the XTT assay using a new type of cell, perform a pre-assay optimization procedure to determine the appropriate XTT Assay conditions.

    

4. Why are absorbance readings too low in wells containing cells and Activated-XTT Solution?
   
   There are a number of potential causes for this problem. The number of cells per well might be too low, the assay incubation time might be too short, the XTT Reagent may have been used without adding the Activation Reagent, or the cells are not growing well. Increase the number of cells per well or incubate the cells for a longer period of time. If you are performing the XTT assay using a new type of cell, perform a pre-assay optimization procedure to determine the appropriate XTT Assay conditions. Prepare Activated-XTT Solution by adding 0.1 mL of the Activation Reagent to 5.0 mL of the XTT Reagent (sufficient for one 96-well microtiter plate assay). If the cells are not growing well, reevaluate and optimize growth conditions or allow more time for post-plating cell recovery before initiating the XTT Assay.

    

5. Why do XTT test replicates have widely varying values?
   
   Inaccurate plating or dispensing of variable reagent volumes can contribute to a lack of assay precision. Ensure the accuracy of your cell counting procedure. Make sure that you are working with a homogenous cell suspension. Check the accuracy of and/or routinely calibrate your precision pipettes.

Ordering

You can order the XTT Cell Proliferation Assay from our online catalog if you have an ATCC account. 


This product is intended for research purposes only. It is not intended for use in humans.