DNA Quantitation Kit – Hoechst Assay
DNA is a reliable endpoint for cell proliferation assays. This proliferation assay, is based on the enhanced fluorescence and shift in the emission wavelength of the fluorochrome Hoechst 33258 upon binding cellular DNA.
At time points of interest, the user have to empty the plates and store them frozen. When the assay is to be performed, cultures are briefly incubated in a lysate media and frozen again. This process lyses the cells and allows rapid and thorough mixing of the fluorochrome and cellular DNA. Freezing permits convenient storage of cultures until the time of assay, then allowing batching experiments. This fact, results in a reduction of the processing time and also gives a better intra and inter-experimental standardization.
The assay here presents, generates a linear standard curve for DNA fluorescence versus cell number. This, enables the rapid and accurate measurement of cell number involving minimal processing time, making this assay suited for cell proliferation studies.
Small quantities of cells can limit tissue culture experiments. Then microcultures are desirable, as they work with primary cell cultures. Therefore, large numbers of test substances, automation, cost efficiency, and statistical analysis can make 96-well microculture plates attractive. An optimal assay for microculture cell quantification would permit the storage of plates so a large number of plates could be assayed at the same time, thus reducing labor. This would also result in better standardization of data within and between experiments. DNA would be a preferable endpoint of cell proliferation and, if possible, the user should measure it without radiolabeled reagents. This optimal assay would require minimal processing time and be highly accurate as a measure of cell proliferation.