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      3. 細胞增殖


        Cell Counting Kit-8 (CCK-8) reagent can be used for simple and accurate Cell proliferation and toxicity analysis.??The basic principle is that the reagent contains WST-8.??2 - (2-4 - nitro phenyl methoxy -) 3 - (4 - nitro phenyl) - 5 - (2, 4 - disulfonic acid benzene) - 2 h - tetrazolium monosodium salt),??It is reduced to a highly water-soluble yellow Formazan dye by dehydrogenase in cells under the action of electron carrier 1-methoxy 5-methylphenazonium dimethyl sulfate (1-methoxy PMS).??The amount of methylzan produced is proportional to the number of living cells.??Therefore, this property can be used directly for cell proliferation and toxicity analysis.??

        1 Methods for CCK8 cell proliferation

        1.1 Preparation of cell suspension: cell counting??

        1.2 Inoculate into 96-well plate: according to the appropriate number of plate cells, about 100ul cell suspension per well, the same sample can be repeated for 3 times. ?

        1.3 Culture in incubator at 37℃ : it takes about 2-4 hours to attach the cells after inoculation. If no need to attach the cells, this step can be omitted. ?

        1.4 Add 10ul CCK8: Because the amount of CCK8 added to each well is relatively small, there may be errors caused by the reagent sticking to the well wall. It is recommended to gently tap the culture plate after adding the reagent to help mix evenly.??Or directly configure the medium containing 10%CCK8, in the form of liquid exchange. ?

        1.5 Culture for 1-4 hours: The amount of Formazan formed varies with different cell types.??If the color rendering is insufficient, culture can be continued to confirm optimal conditions.??In particular, blood cells form very little Formazan and require a long color rendering time (5-6 hours). ?

        1.6 Determination of 450nm absorbance: it is recommended to use dual wavelength for determination, detection wavelength 450-490nm, reference wavelength 600-650nm.??

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