pyrimidinones and diacetylfluorescein

We have recently developed a method for the separation and quantification of viable apoptotic cells without the need for permeabilisation or fixation of the cells. The method is based on the observation that apoptotic rat thymocytes fluoresce more brightly than normal cells after a brief incubation with the DNA binding dye, Hoechst 33342. In order to understand these differences, we have investigated the uptake of Hoechst 33342 by normal and apoptotic thymocytes. By staining with fluorescein diacetate, we have shown that the efflux of fluorescein from apoptotic cells is more rapid than that from normal thymocytes. This result demonstrated an increase in the permeability of the plasma membrane of the apoptotic thymocytes and it is this change which probably results in the more rapid uptake of Hoechst 33342. The data also revealed two populations of apoptotic thymocytes.

Topics: Animals; Apoptosis; Benzimidazoles; Cell Membrane Permeability; Cell Nucleus; Cell Separation; Cells, Cultured; DNA; Flow Cytometry; Fluoresceins; Male; Pyrimidinones; Rats; Rats, Inbred F344; Thymus Gland

Merocyanine 540 (M540) is a potential-sensitive, hydrophobic dye that preferentially incorporates into the 'fluid' domains of cellular membranes, distinguishing between hemopoietic cells according to their differentiation state. A bright staining with M540 is usually achieved by UV illumination of the cells during staining. We show by flow cytometric analysis that: (1) staining is greatly enhanced by UV illumination of mouse spleen cells before addition of the dye; (2) UV treatment causes an increased permeability toward propidium iodide and intracellular fluorescein as well; (3) the increment in M540 fluorescence precedes permeabilization to propidium iodide, while the latter precedes leakage of fluorescein. We also describe an overshoot and accelerated recovery of M540 fluorescence after photobleaching by a 514 nm laser beam. It is suggested that penetration of M540 to the more fluid inner membrane structures explains the fluorescence increment in both experiments.

Topics: Animals; Cell Membrane; Cell Membrane Permeability; Flow Cytometry; Fluoresceins; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence; Photochemistry; Propidium; Pyrimidinones; Radiation-Sensitizing Agents; Rauscher Virus; Spleen; Staining and Labeling; Ultraviolet Rays