In vivo localization of lymphocytes labelled with low concentrations of Hoechst 33342

Hoechst 33342 (HO 33342) is a fluorescent dye which binds specifically to DNA and can be used to label lymphocytes for in vivo migration studies. Lymphocytes were treated with varying concentrations of HO 33342 and assayed in vitro for effects on viability, mitogen-stimulated proliferation, and motility. In vivo traffic studies were performed to determine a dye concentration with minimal toxicity for lymphocytes, but sufficient fluorescence for detection of cells in frozen sections. The concentration reported to yield quantitative staining of nuclear DNA (10.7 microM, or 6 micrograms/ml) reduced motility and proliferative response, and resulted in an altered lymphocyte migration pattern compared to untreated lymphocytes. A concentration of 0.25 microM, however, produced no toxicity in the in vitro assays, and an in vivo migration pattern similar to that of untreated cells; lymphocytes stained with 0.25 microM HO 33342 for 30 min were readily observable in histological sections. This study indicates that the concentration of HO 33342 optimal for DNA staining may exert deleterious effects on in vivo lymphocyte traffic studies, and that far lower dye concentrations are more suitable for such studies.