The determination of cellular viability of hybridoma cells in microtitre plates: a colorimetric assay based on neutral red.

A colorimetric assay utilising Neutral Red (C.I. 50040), a nuclear stain, was developed to determine the cellular viability of hybridoma cells in microtitre plates. A linear correlation (r = 0.99) was found to exist between the uptake of Neutral Red by viable cells and the viable cell count determined by Trypan blue exclusion test. The linearity stretched over the range of cell concentrations normal in batch cultures (2-30 x 10(4)/0.2 ml) with as little as +/- 6% intra-plate well-to-well variation and +/- 10.2% inter-assay variation. Microscopical examinations of viable hybridoma cells stained with Neutral Red showed that it was located in the nucleus. The possible bifunctional activity of the Neutral Red assay as a test for cellular viability and estimating the DNA content of hybridoma cells is discussed along with its application in a drug screening programme.