FLUORESCENCE STAINING OF LIVING CELLS WITH FLUORESCAMINE

The interaction of fluorescamine with living plant and animal cells was investigated to determine which subcellular structures and molecular species might react with the dye and to assess its effects on cell viability and function. Plasma and nuclear membranes of Xenopus erythrocytes, mitochondria of sea urchin sperm, growing apices of Timothy root hairs, and various organelles of Nitella and Euglena were labelled as judged by fluorescence microscopy. Cytoplasmic fluorescence was particulate in Nitella and easily displaced by moderate centrifugal fields in sea urchin eggs. Chloroplasts and nuclei isolated from cells labelled in vivo exhibited fluorescamine dependent fluorescence. Reaction seemed to have little or no effect on cell viability (Euglena) photoautotrophic growth (Euglena), cell motility (sperm), fertilizability (sperm or egg), embryonic development (sea urchin), or cytoplasmic streaming (Nitella, Timothy). Quantitative fluorometric analysis of the in vivo reactants in sperm indicated a reaction preference for phospholipid over protein compared to control cells dissociated in SDS prior to labelling. The bulk of labelled lipid was phosphatidylethanolamine. These results suggest that fluorescamine is a true vital dye which can label the cell surface as well as penetrate deeply within cells to label a variety of organelles. The distribution of fluorescence and results of chemical analysis suggest that in vivo the dye may preferentially react with membrane. © 1979 Springer-Verlag.