The selective detection of viable cells by this method is possible because the proteolytic activity towards the GF-AFC substrate is dependent upon the continued maintenance of membrane integrity. This live-cell, proteolytic activity decays within seconds after a cytotoxic event, so non-viable cells do not contribute appreciably to fluorescence generation. A 30 minute incubation of reagent and cells at 37ºC is typically sufficient for generating a workable signal window, but the incubation can be extended without detriment if work-flow scheduling does not permit shorter periods.