Colorimetric determination of cell numbers by Janus green staining

A colorimetric assay using the basic azo dye Janus green has been developed to assess cell numbers in anchorage-dependent cell cultures, with special regard to the enumeration of osteoblastic cells. Therefore, cells are fixed in ethanol and stained with a 0.2% solution of Janus green for 3 min, foll owed by a destaining step of 1 min in tap water. The addition of diluted hy drochloric acid easily and immediately leads to dye elution from stained ce ll layers into the acidic supernatant which consequently is transferred int o 96-well plates and read on a microplate reader at 595 nm. Working under s tandardized conditions, Janus green uptake in several cell lines is shown t o be linearly correlated with cell numbers over a broad range of cell densi ties, in MC3T3-E1 cells from about 3% up to more than 300% of confluency. A bsolute sensitivity of the assay allows detection of less than 1000 cells/c m(2). In comparison do many other colorimetric assays, the Janus green tech nique is simple to perform, fast, precise, stable, cheap, and well suited f or processing large quantities of samples. Moreover, it is applicable to an y culture formate and size, from irregular formed carriers up to 96-multiwe ll plates. (C) 1999 Academic Press.