A colorimetric microtiter assay for the quantitation of cytokine activity on adherent cells in tissue culture

A colorimetric microtiter assay was developed for the quantitation of adherent cells in culture, which is based on the staining of cells with a commercially available staining kit. Adherent L929, A375 cell lines and human monocytes were stained with Hemacolor reagents and the color eluted with SDS 0.5% was determined spectrophotometrically with an ELISA plate reader at 630 nm. The method enabled the detection of cells and was linear up to 3 × 10⁴ L929 cells/well. The Hemacolor staining assay was compared to the crystal violet staining assay and the MTT reduction assay, and was found to be sensitive, accurate and reproducible, and has the advantage of enabling microscopic inspection of the stained cells prior to color elution. The assay was found to be suitable for the determination of cytotoxic cytokines, and the enumeration of adherent monocytes. This method might be also used for the quantitation of cytotoxic drugs, and the cytophatic activity of viruses.