Cell viability detection kit and preparation method and application thereof

Abstract

The invention relates to a cell viability detection kit and a preparation method and an application thereof. The invention is applicable to live cell counting, and is a determining method for directly detecting the cell proliferation, and is also a detection method for estimating the cell totoxicity of the drugs, peptide growth factors and other substances, and is applicable to detect the suspended cell and the anchorage-dependent cell cultured in the cell culture plates. The invention consists of a WST-8 mixed solution storage solution, a reaction stop solution and a buffer diluent solution, wherein the WST-8 mixed solution storage solution contains 50mmol/L WST-8 solution, 2mmol/L PMS and 1mmol/L PIPES buffer solution; the reaction stop solution is 10%(W/V) sodium dodecyl sulfate; and the buffer diluent solution is 1mmol/L PIPES solution.

Description

technical field [0001] The present invention relates to a cell viability detection kit and its preparation method and application, which are used to count living cells and directly detect cell proliferation, and are also a detection method for evaluating the cytotoxicity of substances such as drugs and polypeptide growth factors. It is suitable for the detection of suspension and adherent cells cultured in cell culture plates. Background technique [0002] The traditional method of cell proliferation detection has been gradually replaced due to radioactive pollution, cumbersome operation, low sensitivity, high requirements for equipment, and high cost. At present, the microenzyme reaction assay of tetramethylazolium salt (MTT) is widely used in the detection of cell proliferation. In the 1960s, scientists discovered that MTT could be reduced to a blue-purple formazan product by succinate dehydrogenase in the mitochondria of living cells, and believed that it had the potenti...

AI Technical Summary

Problems solved by technology

However, the XTT method itself is unstable, relatively insoluble, and requires fresh preparation, and the product of Jiayuezan formed by the XTT method is orange-yellow, and some yellow metabolites and reagents in the culture system can affect the detection results; therefore, a new type of drug is designed. The MTT analog MTS, MTS can be reduced by living cells to form a water-soluble formazan product in the presence of the electron carrier 1-methoxy-5-methylphenazine dimethyl sulfate (1-Methoxy PMS)

Compared with the XTT method, it is easy to operate and has strong specificity. The formed formazan product is dark brown, and the external factors are less affected during detection; 2-(4-iodobenzene)-3-( 4-Nitrophenyl)-5-(2,4-dithiobenzene)-2H-tetrazolium salt (WST-1), also undergoes dehydrogenase reaction in the presence of PMS to form water-soluble formazan product, but WST-1 solution and PMS powder need to be prepared separately, and mixed in proportion when used, and the operation is cumbersome; currently the market recognizes that the best detection sensitivity and ease of operation is 2-(2-methoxy-4 -nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2h-tetrazole monosodium salt (WST-8), a novel tetrazole Azolium salts, like WST-1, require the presence of PMS

With WST-8 as the main component of the reagent configuration, many cell proliferation detection kits based on WST-8 have appeared at home and abroad, but the common problems are 1. The reagent storage period is short, which is easy to cause waste; 2. The detection sensitivity is compared with traditional 3 The H incorporation method needs to be improved; 3. There is no termination reaction solution to terminate the reaction after the color reaction is completed, and the configuration needs to be selected by oneself, which brings inconvenience to the experiment and leads to poor repeatability of detection time