LIGHT AND ELECTRON-MICROSCOPE IMMUNOLABELING AND FLOW CYTOMETRIC EVALUATION OF A 33-KDA STRESS PROTEIN DURING THE CELL-CYCLE OF EUE CELLS GROWN IN HYPERTONIC MEDIUM

The aim of this work was to look for the stress protein P-33, during l ong-term growth of human EUE cells in a hypertonic medium, with a poly clonal antibody against P-33 produced in rabbit, and detectable in cel ls by both light and electron microscopy, using fluorescein-, peroxida se-anti-peroxidase- and gold-indirect immunolabelling. P-33 was found to be mostly located in the cytoplasm, without apparent association wi th subcellular organelles; a slight, but significant, labelling was al so observed in the nucleus. Dual parameter flow cytometric measurement s of fluorescein-immunolabelled P-33 and DNA content (after propidium iodide staining) showed that the amount of P-33 increased progressivel y with increasing times of growth in a hypertonic medium in all phases of the cycle. Therefore there is no direct relationship between the i ncrease of the P-33 and the exit of cells from the cycling compartment that occurs in a hypertonic environment.