Dr. Jace is a research officer in a laboratory that studies anticancer treatment. She is interested to test a plant extract for its apoptosis-inducing effect on colon cancer cells. After incubating the cells with the extracts, she is using the FITC Annexin V Apoptosis Detection Kit I by BD Biosciences for her apoptotic assay. Discuss the detection of apoptosis using this kit and include the utilization of flow cytometry in her experiment.
The maintenance of tissue homeostasis and embryonic development both include the natural physiological process of apoptosis. Certain morphologic characteristics, such as the loss of plasma membrane asymmetry and attachment, condensation of the cytoplasm and nucleus, and DNA internucleosomal cleavage, define the apoptotic programme. One of the early characteristics is the loss of plasma membrane.
Phosphatidylserine (PS), a membrane phospholipid, is translocated from the inner to the outer leaflet of the plasma membrane in apoptotic cells. This exposes PS to the extracellular environment. A 35–36 kDa Ca2+ dependent phospholipid-binding protein called annexin V attaches to cells that have PS exposed and has a high affinity for PS. Annexin V can be coupled to other fluorochromes, such as FITC. When used as a sensitive probe for flow cytometric measurement of cells going through apoptosis, this format maintains its strong affinity for PS. FITC Annexin V labelling can detect apoptosis sooner than assays based on nuclear alterations such DNA fragmentation because externalization of PS occurs in the early stages of apoptosis.
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