产品概述

Proliferation assays are widely used in cell biology for the study of growth factors, cytokines, nutrients and for the screening of cytotoxic or chemotherapeutic agents. There are several ways to determine the number of cells either by microscopic inspection, through an electronic particle counter, indirectly by measuring the incorporation of radioactive precursors, quantifying total protein with chromogenic dyes, or by measuring metabolic activity of cellular enzymes. The most common assay for cell proliferation is the incorporation of 3H‑thymidine into cellular DNA. The 3H-thymidine assay is, however, labor-intensive as it requires the removal of the excess, unincorporated label by using some cell harvesting method before measurement. In 1956, the first paper on the use of tetrazolium salts as indicators of cell viability was published. The method was based on the finding that living cells are capable of reducing slightly colored or uncolored tetrazolium salts into intensely colored formazan derivatives. This reduction process requires functional mitochondria, which are inactivated within a few minutes of cell death. Therefore, this method provides an excellent tool for the discrimination of living and dead cells. However, the early tetrazolium salts did have some disadvantages, such as the insolubility of the resulting formazan products. Time and labor-consuming solubilization procedures were necessary, including repetitive pipetting and mixing, or the application of hazardous solubilizers. This necessary post-assay treatment, however, irreversibly terminated cell proliferation and thus made it impossible to prolong incubation in order to achieve an increase in sensitivity or continue cell culture. These inconve­niences led to the development of non-toxic tetrazolium salts which yield soluble reduction products. Although the assay procedure was made easier by these soluble dyes, in practice the use was limited due to the instability of the formazan dye and a relatively low absorbance of the end product as compared to the classical MTT assay. The Biomedica research department has solved both problems and created an easy-to-use, rapid and reliable non-isotopic cell proliferation assay. For convenience, we have made it highly compatible with the standard thymidine incorporation assay. Therefore, no changes are required in the setup of the test and in the "labeling" procedure. Furthermore, there is no need for the removal of culture medium before or after the addition of the chromogenic substrate and neither solubilization nor harvesting procedures are necessary. The work performed by Biomedica resulted in an assay which combines the best of the thymidine and MTT methods, namely: accuracy, speed, reliability, and ease of use. Also, according to our data achieved so far, the chromophore appears to be non-toxic. A double labeling with EZ4U and a radioactive nucleotide to obtain more information about cell viability and DNA content is now feasible.