THE AMIDO BLACK ASSAY - A SIMPLE AND QUANTITATIVE MULTIPURPOSE TEST OF ADHESION, PROLIFERATION, AND CYTOTOXICITY IN MICROPLATE CULTURES OF KERATINOCYTES (HACAT) AND OTHER CELL-TYPES GROWING ADHERENTLY OR IN SUSPENSION

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 eel types including the human keratinocyte line HaCaT and subclones, mouse 3T3 fibroblasts and myeloma cells X63-Ag8.653. Briefly, cells are fixed by formaldehyde or glutaraldehyde 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 NaOH 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 amido black, 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 c-Ha-ras 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 cytotoxicity of drugs such as hexadecylphosphocholine, target cell killing in co-cultures with interleukin 2-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.