Cell death, overt death or inhibition of growth, occurs by several mechanisms, including necrosis and programmed cell death, most notably apoptosis. Necrosis is generally caused by hostile influences outside the cell designed to kill the cell and is usually initiated by toxic attack from the outside. Apoptosis in normal organism life is caused by environmental influences desiring the removal of the cell in the interest of the overall organism. These influences lead to several mechanisms of eventual death which are preprogrammed and thus the toxic mechanisms are initiated intra-cellularly.
Characterization
Necrosis can be initiated by the host organism, such as Natural Killer (NK) lysis or immune T lymphocytes removing a virus-infected cell, by a toxic agent such as a chemical, Xrays, heat, etc., or by pathogen, or by a therapeutic exercise such as Antibody Dependent Cellular Cytotoxity (ADCC), or cancer chemotoxic drug. The initial direct action can be at the level of the cell membrane or an effect on metabolism, e.g. protein, RNA or DNA synthesis.
Necrosis is characterized by loss of plasma membrane integrity, cellular and organellar swelling, and marked inflammation, leading to disruption of the cellular and nuclear membranes. ATP levels are dramatically reduced in necrotic cells, both because of severe mitochondrial damage that cripples ATP generation as well as unrestrained energy expenditures.
Inhibition of Human Umbillical Endothelial Cell (HUVEC) growth by increasing doses of antibody to Vasculiar Endothelial Cell Growth Factor (VEGF, measured by CellTiter Glo
Apoptosis is characterized by cell shrinkage, DNA cell disintegration, fragmentation into membrane-enclosed apoptotic bodies, and phagocytosis of these corpses by macrophages, or occasionally, neighboring cells. When this clean-up operation is efficient, inflammation is avoided. ATP levels in apoptotic cells are reasonably well maintained both because of continued production and decreased expenditures. The net result of apoptosis is the stealth deletion of individual cells within a tissue.
Methods of Detection
Necrosis assays
Propidium Iodide (PI) assay: Propidium iodide is a fluorescent dye that cannot penetrate intact cell membranes. It is often used in conjunction with flow cytometry or fluorescence microscopy to identify necrotic cells that have compromised membrane integrity.
Annexin V and 7-AAD Assay: This assay uses Annexin V to detect apoptotic cells and 7-AAD to detect necrotic cells with compromised membranes.The combination of these two dyes allows for the discrimination of live, apoptotic, and necrotic cell populations.[
Lactate Dehydrogenase (LDH) Release Assay:
This assay measures the release of the cytoplasmic enzyme LDH into the extracellular medium, which is a marker of necrotic cell death and membrane permeabilization. The LDH released from necrotic cells is quantified using a colorimetric or luminescent readout.
Cell-based assays for the detection of apoptosis
Early stage apoptosis detection assays
Annexin V assay: Annexin V binds to phosphatidylserine, which is exposed on the outer membrane of apoptotic cells. This assay is typically done by flow cytometry.
Mitochondrial membrane potential assay: Early in apoptosis, mitochondrial membranes become permeable, leading to a loss of membrane potential. This can be measured using cell-based assays, flow cytometry, or fluorescent dye that accumulates in the mitochondria.
Cytochrome c release assay: Cytochrome c is released from mitochondria into the cytoplasm during apoptosis, and this can be detected by flow cytometry or fluorescence assays.
Mid-stage apoptosis assays
Caspase activity assays: These measure the activity of caspases, a family of proteases that are activated during apoptosis. Caspase activity can be detected using antibodies or by measuring cleavage of peptide substrates.
Late-stage apoptosis assays
DNA fragmentation assay: Apoptosis leads to characteristic DNA fragmentation, which can be detected by TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) assays.This method provides valuable insight into the apoptotic process by specifically highlighting fragmented DNA.
