EZ4U Cell Proliferation Assay

Produktnummer ABIN5067646

Produktdetails

Reaktivität: Human

Nachweismethode: Colorimetric

Applikation: Cytotoxicity Test (CyTox), Proliferation Assay (ProA)

Verwendungszweck: Non-radioactive cell proliferation and cytotoxicity assay.

Proben: Cell Culture Samples

Spezifität: Cytotoxicity testing

Produktmerkmale: 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, or by the use of an electronic particle counter, indirectly by measuring the incorporation of radioactive precursors, quantitating 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 3Hthymidine assay is, however, labour intensive as it requires the removal of excess, unincorporated label by using some method of cell harvesting before measurement. In 1956, the first paper was published on the use of tetrazolium salts as indicators of cell viability. The method was based on the finding that living cells are capable to reduce slightly or uncoloured tetrazolium salts into intensely coloured formazan derivatives. This reduction process requires functional mitochondria, which are inactivated within a few minutes after cell death. This method therefore provides an excellent tool for the discrimination of living and death cells. However, the early tetrazolium salts did have some disadvantages, such as the insolubility of the resulting formazan products. Time and labour consuming resolubilisation procedures were necessary, including repipetting and mixing, or the application of hazardous solubilisers. 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 "labelling" procedure. Furthermore, there is no need for the removal of culture medium before or after the addition of the chromogenic substrate and neither solubilisation nor harvesting procedures are necessary. The work performed 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 labelling with EZ4U and a radioactive nucleotide to obtain more information about cell viability and DNA content is now feasible.

Bestandteile: activator solution and substrate

Benötigtes Material: pipettes, tubes, reader

Anwendungsinformationen

Probenmenge: 200 μL

Protokoll: All reagents and samples must be at room temperature (18-26 °C) before use in the assay.
Add 200 μL cell culture into respective wells.
Add 20 μL SUB (substrate) into each well, swirl gently.
Incubate at 37 °C for 2-5 hours, depending on the metabolic capacity of the cells.
If no microplate reader with shaking plate carrier is available, mix the plate on a vibrating platform or by tipping with the fingers.
Read absorbance at 450 nm or 492 nm, with 620 nm as reference.
For the most accurate results, absorbance from a substrate blank in assay medium without cells should be subtracted from all other values.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Lagerung: 4 °C