les3 - CHAPTER III: STUDIES OF CELL DEATH AND CELL...

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38 CHAPTER III: STUDIES OF CELL DEATH AND CELL PROLIFERATION III.1. ASSAYS TO MEASURE CELL DEATH (apoptosis – necrosis) III.1.1. Introduction Cells can die in different ways, with necrosis and apoptosis being the best known: Several other types of cell death have been described: pyroptosis, necroptosis, mitotic catastrophe, autophagy, …). These differ mainly in several morphological, biochemical and immunological characteristics. However, the methods in this course will only be discussed for their application to measure cell death by apoptosis or necrosis. Necrosis = accidental cell death Mostly results from physical or chemical stress. Apoptosis = normal or programmed cell death Physiological process to remove surplus of cells or damaged cells during development or other physiological processes. Different diseases are linked to a deregulation of apoptosis (cancer, Alzheimer, AIDS, …). Therefore, interest in signaling mechanisms that contribute to apoptosis has largely increased during recent years. Necrosis and apoptosis can be distinguished both at the morphological level as well as at the biochemical level.
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39 III.1.2. Key elements Membrane changes Protease activity Mitochondrial changes DNA fragmentation
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40 III.1.3. Methods to analyse apoptosis III.1.3.1. Assays based on DNA fragmentation: internucleosomal DNA cleavage by cellular endonucleases results in DNA fragments of specific lengths that can be seen as ' ladders ' in an ethidium bromide stained agarose gel separating these DNA fragments; to increase sensitivity, isolated DNA fragments can be radioactively labeled at their 5’end with T4 polynucleotide kinase and γ -[ 32 P]-ATP. prelabeling (requires dividing cells!!) of DNA with [ 3 H]-thymidine ([ 3 H]-dT) leakage of low molecular weight (LMW) DNA into the cytoplasm of apoptotic cells; determine radioactivity in cytosol via liquid scintillation counting (LSC) prelabeling of DNA (requires dividing cells!!) with the nucleotide analogue 5-bromo-2'- deoxyuridine (BrdU) detection of DNA fragments in cytosol via ELISA (e.g. figure below: microtiter plate with anti-DNA coated wells BrdU labeled DNA microgolf
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41 denaturation anti-BrdU-peroxidase coupled wash + peroxidase substrate spectrophotometric determination of color development). determination of histon-coupled DNA-fragments in cytosol via ELISA: (e.g. figure below: streptavidine-coated well + biotinylated anti-histon antibody + lysate + peroxidase (POD) conjugated anti-DNA wash + peroxidase substrate spectrophotometric determination of color development) Enzymatic labeling of 'nicked' (only 1 broken strand) DNA with labeled nucleotides (e.g. fluorescein-dUTP).
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les3 - CHAPTER III: STUDIES OF CELL DEATH AND CELL...

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