Question

Can you write about the principle of Annexin V/Propidium iodide staining, the principle of Annexin V-FITC binding to PS, and its advantages and disadvantages. Thank you in advance, it really means a lot to me. I appreciate it!!!

Answer 1

Annexin V/Propidium iodide staining are used for cellular apoptosis have been significantly impacted since the introduction of flow cytometry based methods.Propidium iodide(PI) is widely used in conjuction with the help of Annexin V in the determination of cells viability, apoptotic, or necrotic through the differences in plasma member integrity and permiability.The main approach to use the AnnexinV/PI in the study of apoptopic cells.PI used more often than other nuclear strains due to its economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in livining cells.The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptopic cells due to the presence of an intact plasma membrane.In late apoptopic and necrotic cells the integrity of the plasma and nuclear meambranes all decreases, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence.Annexin V/ PI protocols leads to a significant number of false positive events up to 40% which is associated with the PI staining of RNA within the cytoplasmic compartment.Primarly cells and cells lines in a board range of animal models are affected with the large cells, showing the highest occurence.Annexin V/PI method that provides a significant improvement for assesment of cell death compared to the convensional method these all can be demonstrated under such methods.This protocol takes advantage of changes in cellular permiability during cell fixing to promote entry of RNase A cells following staining, it dependes on both timing and concentration and its optimised for the removal of cytoplasmic RNA.

Asymmetric plasma membrane exhibit an healthy cells with phosphatidylserine(PS) located on the cytoplasmic leafleat of the plasma membrane bilayer.Annexin V FITC protien binds PS and its also used for in assess apoptosis in flow cytometry.PS exposed by apoptotic cells serves as a major eat me signalfor phagocytes.Although the exposition of PS has been observed after the alternative stimuli, no clearance of viable, PS exposing cells has been detected.Thus, besides PS exposer , membranes of viable and apoptopic cells might exhibit specific characteristics.Here, we show that Annexin V binds in a cooperative manner to different types of dead cells.The shrinked apoptic cells thereby showed the hghest hill coeffient values.Contrarily, parafaomaldehyde fixation of apoptic cells completely abrogates the cooperative binding of Annexin V to the membranes of apoptic cells reflects higher fluidity of the exposed membranes faciliating PS clustering.

Advantages:-

Its used to evaluate apoptosis in flow cytometry.

Membrane of viable and apoptotic cells might exhibit specific characteristics.

Disadvantages:-

Annexin V binds in a cooperative manner to different types of dead cells

Contrarily, parafomaldehyde fixation of apoptotic cells completely abrogates the cooperatively effects the seen with dad and dying cells.

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