NILE RED LABELING OF SINGLE LIVING CELLS FOR CONTOUR DELINEATION TO QUANTIFY AND EVALUATE THE DISTRIBUTION OF RHODAMINE-123 WITH FLUORESCENCE IMAGE CYTOMETRY

Simultaneous study of intracellular quantification and distribution of fluorescent probes is difficult when cell staining is not homogeneous . This occurs after mitochondrial staining with rhodamine 123 (R123). Classical techniques for evaluation of intracellular R123 fluorescence , such as flow cytometry, are based on measurement of the global fluor escence intensity but do not take into account parameters that reflect ing cellular distribution of the probe. For simultaneously studying in tracellular quantification and distribution of R123 with fluorescence image analysis, we delineated a mask of the cell, generated from a flu orescent image of the plasma membrane stained by nile red (NR). After a preliminary study of the fluorescence characteristics of R123 and NR to avoid artifacts and optimize conditions of staining, quantificatio n and distribution of intracellular R123 studies were performed by sup erimposition of the mask on the R123 fluorescence image. This protocol was applied to leukemic cells and allowed estimation of individual ce ll parameters such as mean fluorescence intensity and standard deviati on, the latter providing information of the cellular distribution ofR1 23. Moreover, it permitted demonstration of the redistribution of R123 in the whole cell when coincubated in the presence of nigericin.