Core A: Cell and Tissue Pathology will provide endpoint analyses for the study of cell death in beth cell culture systems and in animal and human tissues. Endpoint analyses for cell culture include brightfield examination (LM) using Romanowsky-type dyes or polychrome stains, transmission electron microscopy (TEM), agarose gel electrophoresis, nucleosomal ELISA assay and flow cytometry. The non-morphological assays for apoptosis will be confirmed by morphology and will be used as apoptosis endpoints if they accurately measure apoptosis. Apoptosis will be quantitated in animal and human colonic specimens using LM. The in situ DNA fragmentation assays will be used as a measure of DNA damage in the cell culture experiments. The tailing method using TdT and the nick translation methods using DNA polymerase I (Klenow fragment) and T7 polymerase in the presence of digoxigenin-labelled nucleotides will be compared for sensitivity to DNA damage and evaluated using routine fluorescence and laser scanning confocal microscopy (LSCM). These assays will be compared with morphology to indicate if the DNA damage precedes or is part of the apoptosis process itself. The live cell/dead cell fluorescent assay, which uses acridine orange and ethidiumbromide, will be used to evaluate toxicity levels of drugs, since apoptotic and necrotic cells can be simultaneously evaluated using routine fluorescence microscopy. The core is also designed to develop special microscopic procedures to answer specific questions that emerge as experimental data is obtained and analyzed. A live cell set-up will be added to the Leica LSCM within the next 2 months, which will allow investigators to get a high resolution image in the z-direction for cellular localization of oxidative stress fluorochromes. High-resolution line-filtered images of fluorochrome-loaded cells before and after the addition of an apoptosis-inducing agent can be obtained, digitized and analyzed using semi-quantitative analysis. Since a major emphasis of this PPG is on experimentation with cell culture, the use of fluorochrome tags on secondary antibodies used to detect bcl-2, catalase, NFkappaB, PCNA and in situ DNA fragmentation, in conjunction with LSCM, will be explored. Other major responsibilities of this core are to quality control the """"""""resistance-to-apoptosis"""""""" bioassay, provide pathologic evaluations of selected animal and human tissues and interact with the on- going clinical trials.
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