XTT - Cell Proliferation Kit

Cell Proliferation Kit (XTT Based)
Cat. # Product Name Unit Size Storage Temp.
20-300-1000 XTT Kit 1000 assays -20°C
 

Introduction

Cell proliferation assays are w idely used in cell biology for the study of grow th factors, cytokines and media components, for the screening of cytotoxic agents and for lymphocyte activation.
The need for a reliable, sensitive and quantitative assay that w ould enable analysis of a large number of samples led to the development of methods, such as:
  • Use of radioactive thymidine to label DNA in live cells
  • Use of Brdu to label DNA in live cells (as a substitute for radioactive thymidine)

The above methods have a number of disadvantages, including: use of radioactive materials and relatively complex techniques. The use of tetrazolium salts, such as MTT, commenced in the 1950s and is based on the fact that live cells reduce tetrazolium salts into colored formazan compounds. The biochemical procedure is based on the activity of mitochondria enzymes w hich are inactivated shortly after cell death. This method w as found to be very efficient in assessing the viability of cells. A colorimetric method based on the tetrazolium salt, XTT, w as first described by P.A. Scudiero in 1988. Whilst the use of MTT produced a non-soluble formazan compound w hich necessitated dissolving the dye in order to measure it, the use of XTT produces a soluble dye. The use of XTT greatly simplifies the procedure of measuring proliferation, and is, therefore, an excellent solution to the quantitating of cells and their viability w ithout using radioactive isotopes. This kit w as developed to assay cell proliferation in reaction to different grow th factors, cytokines and nutrient components. In addition, it is suitable for assaying cytotoxicity of materials such as TNF or other grow th inhibitors. XTT can be used as a non-radioactive substitute for cytotoxic tests based on the release of 51Cr from cells w ith no less sensitivity.

The advantages of using this kit can be summarized w ith the follow ing attributes:
  • easy-to-use: there is no requirement for additional reagents and/or the cell w ashing procedures
  • speed: multiw ell plates and an ELISA reader can be used for reading
  • sensitivity: can be assayed even in low cell concentrations
  • accuracy: dye absorbance is proportional to the number of cells in each w ell
  • safety: there is no need for radioactive isotopes

Kit Components
XTT Reagent (10x5ml)
A sterile solution containing the XTT reagent. The solution should be stored frozen and should not be exposed to light. To avoid repeated re-freezing, dividing the solution into a number of vials after defrosting the original vial is recommended. Note: if sediment is present in the solution, heat the solution to 37°C and swirl gently until a clear solution is obtained.

Activation Reagent (2x0.5ml)
A sterile solution containing PMS (N-methyl dibenzopyrazine methyl sulfate). The solution should be stored frozen and should not be exposed to light. To avoid repeated re-freezing, dividing the solution into a number of vials after defrosting the original vial is recommended.
Note: if sediment is present in the solution, heat the solution to 37°C and swirl gently until a clear solution is obtained.

Assay Principles
The assay is based on the ability of metabolic active cells to reduce the tetrazolium salt XTT to orange colored compounds of formazan. The dye formed is w ater soluble and the dye intensity can be read at a given w avelength w ith a spectrophotometer. The intensity of the dye is proportional to the number of metabolic active cells. The use of multiw ell plates and an ELISA reader enables testing a large number of samples and obtaining easy and rapid results. The test procedure includes cultivation of cells in a 96-w ell plate, adding the XTT reagent and incubation for 2-24 hours. During incubation an orange color is formed, the intensity of w hich can be measured w ith a spectrophotometer, in this instance w ith an ELISA reader. The greater the number of active cells in the w ell, the greater the activity of mitochondria enzymes, and the higher the concentration of the dye formed, w hich can then be measured and quantitated.

Procedure
  • The cells should be cultivated in a flat 96-w ell plate. To each w ell add 100μl of grow th media. The cells should be incubated in a CO2 incubator at 37°C. In most cases cells can be used to assay proliferation after 24-96 hours.
  • Defrost the XTT reagent solution and the activation solution immediately prior to use in a 37°C bath. Sw irl gently until clear solutions are obtained.
  • To prepare a reaction solution sufficient for one plate (96 w ells), add 0.1ml activation solution to 5ml XTT reagent.
  • Add 50μl of the reaction solution to each w ell and incubate the plate in an incubator for 2-24 hours (usually, 2-5 hours are sufficient).
  • Shake the plate gently to evenly distribute the dye in the w ells.
  • Measure the absorbance of the samples w ith a spectrophotometer (ELISA reader) at a w avelength of 450-500 nanometer. In order to measure reference absorbance (to measure non-specific readings), use a w avelength of 630-690 nanometer.

Notes
  • Defrost and prepare the reaction mixture only immediately prior to use.
  • Since the test is extremely sensitive, it is possible to use a low concentration of cells in the w ells (approximately 5000 cells per w ell). Since there are cell types w hich show low metabolic activity, such as lymphocytes, kartinocytes and melanocytes, it is recommended to increase the concentration of cells to 2.5x105 cells per w ell, in order to obtain development of formazan color w ithin a reasonable period of time.
  • Incubation time w ith the reaction mixture varies according to the type and concentration of the cells. Therefore, it is advisable to perform an initial test by reading the absorbance at various time lapses, i.e. after 4, 6, 8, 12 hours using the same plate.
  • Prior to reading the absorbance w ith a spectrophotometer, the plate should be gently shaken in order to evenly distribute the dye in the w ells.
  • If the volume of the media in each w ell is larger than 100μl, add a larger amount of reaction mixture by the same increment (i.e. 100μl reaction mixture to 200μl grow th media).

Summary of Proliferation Assay Using XTT Reagent: Flow Chart
  • Defrost XTT reagent and activation reagent (37°C)
  • Prepare reaction mixture (0.1ml activation reagent and 5ml XTT reagent for one plate)
  • Add 50μl reaction mixture for each w ell (containing 100μl media)
  • Incubate at 37°C for 2-24 hours (in most cases incubation for 2-5 hours is sufficient)
  • Measure absorbance at a w avelength of 450-500 nanometer (reference absorbance: at a w avelength of 630- 690 nanometer)

References
  • Hansen, M.B., et al, (1989), J. Immunol. Meth. 119, 203-210
  • Jost, L.M., et al, (1992), J. Immunol. Meth. 147, 153-165
  • Roehm, N.W., et al, (1991), J. Immunol. Meth. 142, 257-265
  • Scudiero, P.A., et al, (1988), Cancer Res. 48, 4827-4833
  • Tada, H., et al, (1986), J. Immunol. Meth. 93, 157-165
  • Weislow, O.S., et al, (1989), J. Natl Cancer Inst. 81, 577-586