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The fluorescent stain Hoechst 33342, when injected i.v. into mice, has an LD50 of 300 micrograms g-1. The stain exits rapidly from the blood, with a half-life of 110 sec following an injection of 10 micrograms g-1, but remains bound within target cells, redistributing with a half-life longer than 2 h. This results in a gradient of drug binding outward from capillaries which can be used to estimate regional perfusion via fluorescence microscopy of frozen tissue sections. For tumour tissues that can be dispersed into single cell suspensions, intracellular Hoeschst 33342 can be quantified by flow cytometry, and cell populations can be selected on the basis of their fluorescence (distance from the vasculature) using a fluorescence-activated cell sorter. Our results in tumours and in spheroids indicate that the rate of stain uptake by different cell subpopulations in situ is much more dependent on stain delivery than on selective uptake. Retention of the stain in spheroids is sufficiently stable to allow cell sorting several hours post-injection. Hoechst 33342 thus appears to have considerable potential as an agent for quantifying tissue perfusion, and for allowing selection of tumour cell subpopulations to assess response to radiation and drugs.

Pharmacokinetics, binding and distribution of Hoechst 33342 in spheroids and murine tumours