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.

摘要: A new multipurpose cell micro-assay has been developed, using the protein dye amido black 10B as an indicator of numbers in 96-well plates. The assay is reliable, rapidly performed and can be combined with morphological evaluation photography stained cells. It permits investigations various types including human keratinocyte line HaCaT subclones, mouse 3T3 fibroblasts myeloma cells X63-Ag8.653. Briefly, are fixed by formaldehyde or glutaraldehyde and, following aspiration fixative non-adherent cells, at pH 3.5. protein-bound completely eluted NaOH scanned a microplate reader 620 nm against 405 750 nm. Non-adherent semi-adherent assayed centrifugation plates before fixation. revealed good linear correlation between absorbance black, count DNA content within range 1000-64,000 cells/well. slope regression varied different types. Experiments its c-Ha-ras oncogene-transfected subclones demonstrated suitability for optimizing culture conditions, dose-response studies screening quantification adhesion to extracellular matrix molecules. was also used evaluate cytotoxicity drugs such hexadecylphosphocholine, target killing co-cultures interleukin-2-activated lymphocytes, testing hybridoma antibodies their biological effects on proliferation adhesion. highly reproducible, sensitive, independent cellular aggregation economic multiple applications.