The amido black assay: a simple and quantitative multipurpose test of adhesion, proliferation, and XXXX in microplate cultures of keratinocytes (HaCaT) and other cell types growing adherently or in suspension.

@entity1 @entity14219 @entity137 @entity11517 @entity19 @entity2047 @entity3584 @entity406 @entity1582 @entity71325 @entity6674
A new multipurpose cell micro-assay has been developed, using the protein dye amido black 10B as an indicator of cell numbers in 96-well plates. The assay is reliable, rapidly performed and can be combined with morphological evaluation and photography of stained cells. It permits investigations of various cell types including the @entity1 keratinocyte line HaCaT and subclones, @entity19 3T3 fibroblasts and @entity1582 cells @entity19 . Briefly, cells are fixed by @entity406 or @entity3584 and, following aspiration of fixative and non-adherent cells, are stained by amido black at pH 3.5. The protein-bound dye is completely eluted by @entity6674 and is scanned in a microplate reader at 620 nm against 405 nm or 750 nm. Non-adherent and semi-adherent cells are assayed by centrifugation of plates before fixation. The assay revealed a good linear correlation between absorbance of @entity71325 , cell count and DNA content within the range 1000-64,000 HaCaT cells/well. The slope of the regression line varied with different cell types. Experiments with HaCaT cells and its @entity11517 oncogene-transfected subclones demonstrated the suitability of the assay for optimizing culture conditions, dose-response studies and for the screening and quantification of cell adhesion to extracellular matrix molecules. The assay was also used to evaluate @entity137 of drugs such as @entity14219 , target cell killing in co-cultures with @entity2047 -activated lymphocytes, and the testing of hybridoma antibodies for their biological effects on proliferation and adhesion. The assay is highly reproducible, sensitive, independent of cellular aggregation and economic for multiple applications.
