Apoptosis Lab Report

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The term Apoptosis was first used by scientists Kerr, Wyllie and Currie in the year 1972. The term “apoptosis” has a Greek origin and means “falling off.” Apoptosis or programmed cell death, can be described as an active process that is seen in multicellular organisms. It is characterized by the activation of biochemical pathways that lead to changes in cell morphology. These morphological changes include: cell shrinkage, DNA fragmentation, chromatin condensation and formation of apoptotic bodies. Changes such as mitochondrial breakdown to release cytochrome c and the translocation of phosphatidylserine from the inner plasma membrane leaflet to the outer leaflet also occur. The changes that occur in the cell, act as signals of
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There should be a control on the number of cells that are killed by apoptosis as a high rate of programmed cell death will result in atrophy. On the other hand a rate of apoptosis that is lower than usual, will result in too many cells and may lead to uncontrolled cell proliferation (cancer).
The structural changes that occur during apoptosis can be described in two stages:
1. Nuclear and cytoplasmic condensation occurs. The cell breaks up into small membrane bound bodies known as apoptotic bodies.
2. The apoptotic bodies are taken up by phagocytes and are degraded.

In a healthy adult human, billions of cells die in the bone morrow and intestine every hour. In some cases apoptosis helps regulate cell number. The apoptotic bodies produced from dying or injured cells (due to damage or toxins) are quickly removed before they can spill out their cellular contents. This avoids inflammation of surrounding
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Changes in cell morphology: morphological changes such as cell shrinkage, membrane blebbing, nuclear condensation, DNA fragmentation and changes in plasma membrane occur. Each of these changes can be quantified using Flow Cytometry. For example, the forward scatter parameter reduces on cell shrinkage while nuclear condensation causes an increase in side scatter.
Apoptotic cells can also be detected using hematoxylin and eosin staining, using light microscopy. Although this is a simple technique, it cannot detect apoptotic cells in early stages and the technique needs to be supplemented with other methods of detection. Transmission Electron Microscopy (TEM) is considered as the gold standard to confirm apoptosis. TEM can detect apoptotic bodies, phagocytosis of apoptotic bodies and nuclear fragmentation among other changes. The main disadvantages of this method include the cost, time required for detection and the low throughput.

2. Membrane alterations: translocation of phosphatidylserine (PS) occurs during apoptosis. Exposure of PS on the outer leaflet of the plasma membrane can be detected using Annexin V. Annexin V binds to the PS and is in turn tagged with a label such as phycoerythrin or fluorescein isothiocyanate (FITC). These labels can be detected using flow Cytometry.
Alterations in membrane permeability can be detected using 7-aminoactinomycin

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