Spectrophotometric quantitation of anchorage-dependent cell numbers using extraction of naphthol blue-black-stained...

A convenient method is described by which the actual or relative number of cells in anchorage culture is determined. After removal of the growth medium, cells are subjected to a double-fixation procedure. The cellular protein content is subsequently quantitatively stained with naphthol blue-black. After a period of removal of unbound stain, dye-protein complexes are hydrolytically released and measured spectrophotometrically at 620 nm. A linear correlation exists (r = 0.994) between cell concentration, in the range 3 X 10(4) to 8 X 10(5) cells/ml of final assay volume, and absorbance up to reading values of 3.8. The technical reproducibility of the assay, as judged from assessments of cell numbers in suspension culture, displays a coefficient of variation of 5%. The method was developed for 9.6-cm2 culture dishes, but it should be possible to transform it for the use of microtiter plates.