Factors affecting the reproducibility of a spectrofluorimetric assay for the enumeration of human venous endothelium in culture.

In an EDTA/Hoechst 33258 assay system, a linear increase in fluorescence with increase in cell number between 2 X 10(3) and 1 X 10(5) was obtained if a dye concentration of 800 ng/ml was used. For a given number of cells, the enhancement of fluorescence was found to be greater than that of a theoretically equivalent of DNA. A standard curve for the assay was derived by plotting enhancement of fluorescence against cell number. The effect of storage on the fluorescence of intact monolayers, cellular or commercial DNA, or dye-DNA complexes made it essential that the assay was carried out on fresh samples.