What happens in the sub G1 phase?
One of the most widely used assays to determine apoptosis by flow cytometry is the estimation of fractional DNA content (aka sub-G1 assay). During apoptosis, genomic DNA is cleaved into smaller fragments, each approximately 180 bp (and multiples of it). It is the Sub-G1 peak.
What is SubG1?
UCL Institute of Child Health. 30 Guilford Street, London WC1N 1EH. Tel 020 7405 9200 ext 0198. SubG1 Analysis Using Propidium Iodide. Background.
What is flow cytometry in apoptosis?
Flow cytometry is one of the most popular and versatile applications for studying apoptosis. Flow cytometry allows the study of all aspects of apoptosis from induction via surface receptors, to late stages where DNA fragmentation occurs.
How does flow cytometry detect apoptosis?
One of the classical flow cytometric methods to detect apoptosis is using annexin V binding to phosphatidylserine residues normally located within the plasma membrane. Phosphotidylserine residues are externalised during apoptosis, so only cells that have decided to die will be detected by annexin V binding.
What is sub G1 population?
In general the sub-G1 “population” is comprised of cell fragments, often as a result of apoptosis, however you may also find aneuploidic cells in here too, it depends on how “sub” the population is.
How is DNA fragmented?
DNA fragmentation is a biochemical hallmark of apoptosis. In dying cells, DNA is cleaved by an endonuclease that fragments the chromatin into nucleosomal units, which are multiples of about 180-bp oligomers and appear as a DNA ladder when run on an agarose gel.
What is a necrotic cell?
Necrosis (from Ancient Greek νέκρωσις, nékrōsis, “death”) is a form of cell injury which results in the premature death of cells in living tissue by autolysis. Necrosis is caused by factors external to the cell or tissue, such as infection, or trauma which result in the unregulated digestion of cell components.
How does flow cytometry measure cell death?
Flow cytometry is a valuable tool for the simultaneous assessment of necrosis and apoptosis in a single population of cells. Necrosis is detected by measuring the permeability of the plasma membrane to a normally impermeable fluorescent dye, such as the DNA-binding dye propidium iodide (PI).
How does propidium iodide stain dead cells?
Propidium Iodide (PI) is an intercalating fluorescent agent that binds between the bases of DNA. Propidium Iodide is membrane impermeant, which prevents DNA binding in viable cells, allowing identification of dead cells in a population. PI is an intercalating red fluorescent agent that binds between the bases of DNA.
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