MTT原理及应用

For life science research only. Not for use in diagnostic procedures. FOR IN VITRO USE ONLY.

Cell Proliferation Kit I (MTT)

Colorimetric assay (MTT based) for the non-radioactive quantification of cell proliferation and viability

Cat. No. 1 465 0071 Kit (for 2500 tests)

Introduction

The determination of cellular proliferation, viability and activation are key areas in a wide variety of cell biolog-ical approaches. The need for sensitive, quantitative, reliable and automated methods led to the develop-ment of standard assays. Such an example is based on the capability of the cells to incorporate a radioactively labeled substance ([3H])- thymidine), or to release a radioisotope such as [51Cr] after cell lysis. Alternatively, the incorporation of 5-bromo-2’-deoxyuridine (BrdU)* in place of thymidine is monitored as a parameter for DNA synthesis and cellular proliferation in immuno-histo- and cytochemistry, in a cell ELISA and FACS analysis. (kits and reagents for these applications are available from Roche Molecular Biochemicals).

Cell proliferation and viability assays are of particular importance for routine applications. Tetrazolium salts (e.g. MTT, XTT, WST-1) are especially useful for assaying the quantification of viable cells, because they are cleaved to form a formazan dye (fig. 1; for UV absor-bance spectrum, see fig. 2) only by metabolic active cells.

Fig.1: Metabolization of MTT to a formazan salt by viable cells.

ecnabrobaFig. 2: Comparison of UV-spectra of MTT labeling reagent (dotted line) and the formazan salt after solubilization with solubilization solution.

0200.C72.2.1465 503 R CB GPM

Version 3, October 1999

Store at 15 to 25°C

The non-radioactive, colorimetric assay system using MTT was first described by Mosmann, T. et al. (1) and improved in subsequent years by several other investi-gators (2–6). The assay is designed for the spectro-photometric quantification of cell growth and viability (1, 3, 5–7) without the use of radioactive isotopes. It is used for the measurement of cell proliferation in response to growth factors, cytokines and nutrients (1–3, 6, 8–12) (see fig. 4).

The MTT assay is also useful for the measurement of cytotoxicity. Examples are the quantification of tumor necrosis factor-a or -b effects (13, 14). (see fig. 5) or macrophage induced cell death (15, 16) and the assessment of cytotoxic (17–34) or growth inhibiting agents such as inhibitory antibodies (see fig. 6).

For the replacement of the radioactive [51Cr]-release cytotoxicity assay, protocols using MTT have been developed. The MTT assay is as sensitive as the radio-active method, but shows a significantly lower back-ground especially after long term incubation (34). The MTT assay can also be used to study cell activation (4). Compared to radioactive isotope techniques, the Cell Proliferation Kit I (MTT) is safer-no radioactive isotopes are used, accurate-the absorbance revealed, strongly

correlates to the cell number, (see fig. 3)

sensitive-low cell numbers are detected (see fig. 3). fast-the use of multiwell-ELISA readers

allows for processing a large number of samples,

easy-no washing steps and no additional

reagents are required,

Product description

The kit contains

1. MTT labeling reagent, (1×, ready-to-use)

5 vials containing 5 ml MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid) labeling reagent (1x), 5 mg/ml, in phosphate buffered saline (PBS), non-sterile, ready to use.

Stability

Stable at 15 to 25°C protected from light. Repeated thaw-freeze cycles do not affect product stability. After thawing, the MTT labeling reagents may be stored pro-tected from light at 2-8°C for up to 4 weeks, in which case a sterile filtration of the reagent is recommended.

2. Solubilization solution, (1×, ready-to-use)

3 bottles solubilization solution (90 ml per bottle), 10% SDS in 0.01 M HCI.

Stability

Stable at 15-25°C.