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Detecting Apoptosis: Methods for Analyzing Programmed Cell Death
- 2. LOGO — 2 — Introduction Part 1 Mechanism Part 2 Disease Part 3 Detection Part 4 Creative Bioarray CONTENTS — 2 —
- 3. Part 1 Introduction — 3 — Creative Bioarray Apoptosis removes cells during development, eliminates potentially cancerous and virus-infected cells, and maintains balance in the body. Apoptosis is a form of programmed cell death, or “cellular suicide.” Apoptosis is an orderly process in which the cell’s contents are packaged into small packets of membrane for “garbage collection” by immune cells.
- 4. 余世维 Morphology of Apoptosis Morphologically, apoptosis is first characterized by a change in the refractive index of the cell followed by cytoplasmic shrinkage and nuclear condensation. The cell membrane begins to show blebs or spikes (protrusions of the cell membrane), depending on cell type and eventually these blebs and spikes separate from the dying cell and form "apoptotic bodies". — 4 — Creative Bioarray Part 1 Introduction
- 5. LOGO — 5 — Apoptosis VS Necrosis • Apoptosis • Necrosis • Apoptosis VS Necrosis Creative Bioarray Part 1 Introduction — 5 —
- 6. 余世维 Apoptosis Pathway of cell death induced by a tightly regulated suicide program. Controlled by specific genes. Fragmentation of DNA. Fragmentation of nucleus. Blebs form and apoptotic bodies are released. Apoptotic bodies are phagocytized. No neutrophils. — 6 — Creative Bioarray Part 1 Introduction
- 7. 余世维 Necrosis Morphologic expression of cell death. Progressive disintegration of cell structure. Initiated by overwhelming stress. Usually elicits an acute inflammatory cell response (neutrophils may be present). — 7 — Creative Bioarray Part 1 Introduction
- 8. — 8 —Introduction Creative Bioarray Part 1 Comparison of morphological features of apoptosis and necrosis Apoptosis (programmed cell suicide) Necrosis (uncontrolled cell death) Size Cellular shrinkage Cellular swelling One cell affected Many cells affected Uptake Cell contents ingested by neighbouring cells Cell contents ingested by macrophages No inflammatory response Significant inflammation Membrane Membrane blebbing, but integrity maintained Loss of membrane integrity Apoptotic bodies form Cell lysis occurs Organelles Mitochondria release pro-apoptotic proteins Organelle swelling and lysosomal leakage Chromatin condensation and non- random DNA degradation Random degradation of DNA
- 9. — 9 — Creative Bioarray Part 2 Mechanism There are two major types of apoptosis pathways, each of which illustrates an important point about how apoptosis is triggered and why it is useful. Apoptosis pathway
- 10. — 10 — Creative Bioarray Part 2 Mechanism In the extrinsic pathway, a signal is received from outside the cell instructing it to commit programmed cell death. This may occur if the cell is no longer needed, or if it is diseased. Extrinsic pathway Like many pathways for bringing about complex changes in a cell, the extrinsic pathway to apoptosis involves many steps, each of which can be up-regulated or down- regulated by gene expression or by other molecules.
- 11. — 11 — Creative Bioarray Part 2 Mechanism The intrinsic pathway is triggered by stress or damage, which can lead the cell to apoptosis include damage to its DNA, oxygen deprivation, and other stresses that impair a cell’s ability to function. Intrinsic pathway In response to these damages or stresses, the cell “decides” that its continued existence might be dangerous or costly to the organism as a whole. It then activates a set of proteins called “BH3-only proteins.”
- 12. — 12 — Creative Bioarray Part 3 Disease In some cases, a cell can pose a threat to the rest of the body if it survives, which may be the case for cells with DNA damage, pre-cancerous cells, and cells infected by viruses. When a cell’s DNA is damaged, it will typically detect the damage and try to repair it. If the damage is beyond repair, the cell will normally send itself into apoptosis. When cells have DNA damage but fail to undergo apoptosis, they may be on the road to cancer. Sometimes, pre-cancerous cells manage to duck both internal and external cues that would normally trigger apoptosis. This allows them to divide out of control and accumulate mutations (changes in their DNA). Apoptosis can eliminate infected or cancerous cells
- 13. LOGO — 13 — Apoptosis Detection • TUNEL • Agarose Gel Electrophoresis • Cytochrome C Release Creative Bioarray Part 4 Detection Methods — 13 — • Plasma Membrane Alterations • Caspase Activation • Mitochondrial Changes
- 14. — 14 — Creative Bioarray Part 4 Detection Methods Plasma Membrane alterations Annexin V Apoptosis Assay1 Annexin V apoptosis assay is based on the measurement of the loss of plasma membrane asymmetry. Analysis is typically by flow cytometry. Annexin V binds to phosphatidylserine, which migrates to the outer plasma membrane in apoptosis. Pair Annexin V with a membrane impermeable dye like PI to distinguish between intact, apoptotic, and necrotic cells.
- 15. — 15 — Creative Bioarray Part 4 Detection Methods Caspase Activation Caspase Activity Determination2 Caspases are synthesized as inactive precursors, which are activated by proteolytic cleavage to generate active enzymes. The activation of caspases is a common and critical regulator of the execution phase of apoptosis, triggered by many factors, including treatment with radio- and chemotheraputic agents. Activation of caspases can be examined through a variety of methods: colorimetric, immunoblot, or immunohistochemical.
- 16. — 16 — Creative Bioarray Part 4 Detection Methods Mitochondrial Changes Mitochondrial Membrane Potential Assay3 Mitochondrial membrane potential (MMP) is an important parameter of mitochondrial function that has been used as an indicator of cell apoptosis. Several cell membrane permeable fluorescent dyes, such as rhodamine-123 (Rh-123), 3, 3′- dihexyloxacarbocyanine iodide (DiOC6), 5,5’,6,6’- tetrachloro-1,1’,3,3’-tetraethylbenzimi- dazolylcarbocyanine iodide (JC-1), tetramethyl rhodamine methyl and ethyl esters (TMRM and TMRE), are currently available to measure changes in MMP.
- 17. — 17 — Creative Bioarray Part 4 Detection Methods Mitochondrial Changes Cytochrome C Release4 Mitochondrial cytochrome C has been found to have dual functions in controlling both cellular energetic metabolism and apoptosis. Through interaction with apoptotic protease activating factors (Apaf), cytochrome C can initiate the activation cascade of caspases once it is released into the cytosol.
- 18. — 18 — Creative Bioarray Part 4 Detection Methods DNA Fragmentation TUNEL5 A hallmark of late apoptosis is extensive genomic DNA fragmentation that generates a multitude of DNA double-strand breaks (DSBs) with accessible 3'-hydroxyl (3'-OH) groups. TUNEL assays identify apoptotic cells by the terminal deoxynucleotidyl transferase (TdT)- mediated addition of labeled (X) deoxyuridine triphosphate nucleotides (X-dUTPs) to the 3’- OH end of DNA strand breaks that are subsequently visualized depending on the introduced.
- 19. — 19 — Creative Bioarray Part 4 Detection Methods DNA Fragmentation Agarose Gel Electrophoresis6 Apoptosis is associated with the fragmentation of chromosomal DNA into multiples of the 180- 200 bp nucleosomal unit, known as DNA laddering. In DNA laddering assay, small fragments of oligonucleosomal DNA is extracted selectively from the cells whereas the higher molecular weight DNA stays associated with the nuclei. The isolated DNA is separated by electrophoresis and visualized using ethidium bromide.
- 20. — 20 — Creative Bioarray The End
- 21. LOGO — 21 — Add ress Tel Fax info@creative-bioarray.com 1-631-626-9181 1-631-614-7828 45-1 Ramsey Road, Shirley, NY 11967, USA E- mail