New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy

Abstract

16 fluorochromes were examined for their ability to label viable lymphocytes in vitro and yield fluorescence detectable by fluorescence microscopy and flow cytometry. Of these fluorochromes, four intracellular dyes were found to be suitable for in vivo migration studies. They were H33342, the well known DNA-binding dye which excites and emits in the UV range, and three fluorescein based cytoplasmic dyes, namely BCECF-AM, Calcein-AM and CFSE which excite and emit in the visible range. Lymphocytes labelled with H33342, BCECF-AM and Calcein-AM were suitable for short term in vivo migration experiments with detection by flow cytometry 2-3 days post injection. In contrast lymphocytes labelled with CFSE, a fluorochrome which can covalently couple with intracellular macromolecules, were detected by flow cytometry up to 8 weeks post injection and thus this fluorochrome is ideal for long term migration experiments. Due to marked differences in fluorescence profiles, BCECF-AM and Calcein-AM could be used for short term double labelling experiments using the flow cytometer in which entry of injected lymphocytes into lymphoid organs was quantified. Similarly, in vivo localization of lymphocyte subpopulations could be examined by fluorescence microscopy utilizing differences in fluorescence excitation and emission spectra of lymphocytes labelled with H33342 and one of the fluorescein based dyes.