Flow cytometric analysis of the uptake of Hoechst 33342 dye by human lymphocytes

Human peripheral blood lymphocytes have been shown to resist staining with the DNA binding fluorochrome Hoechst 33342 by the cellular membrane. The rate of uptake of the dye is strongly temperature-dependent with minimal uptake rate below 16 degrees C. The activation energy of dye transport was found to be 135 kJ/mol above 20 degrees C and about 20 kJ/mol below 16 degrees C. Metabolic inhibitors accelerated, instead of inhibiting, the transport of the dye. Dead cells have been shown to stain promptly in contrast with the gradually staining viable cells. The uptake process in the vital staining conditions is suggested to involve a carrier mediated mechanism. Application of Hoechst 33342 as a fluorescent indicator of viability is proposed.