What does annexin V do?
Annexin A5 (or annexin V) is a cellular protein in the annexin group. In flow cytometry, annexin V is commonly used to detect apoptotic cells by its ability to bind to phosphatidylserine, a marker of apoptosis when it is on the outer leaflet of the plasma membrane.
What is annexin V flow cytometry?
Annexin-V is a specific PS-binding protein that can be used to detect apoptotic cells. The Millipore Muse mini flow cytometer can be used to determine apoptosis levels in a cell sample by labelling with annexin V-PE and 7-AAD as the viability dye, see figure.
How does annexin V assay work?
Assay Principle: Annexin V binds specifically to phosphatidylserine and labelled Annexin V can be used detect apoptotic cells. Binding of Annexin V to the exposed charged head groups of PS is a Ca2+ dependent process. Propidium Iodide for control staining of necrotic cells is also available.
What is Annexin PI assay?
The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells3. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases7,8, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence 1,2,9.
How does annexin V detect apoptosis?
Why is annexin V staining used to detect apoptosis? Fluorescent conjugates of annexin V are commonly used to identify apoptotic cells. It has been reported that the translocated phosphatidylserine on the outer surface of the cell marks the cell for recognition and phagocytosis by macrophages (1).
What is the principle of analysis of apoptotic cells through flow cytometry upon staining with annexin V propidium iodide staining?
It is based on the principle that normal cells are hydrophobic in nature as they express phosphatidyl serine in the inner membrane (side facing the cytoplasm) and when the cells undergo apoptosis, the inner membrane flips to become the outer membrane, thus exposing phosphatidyl serine.
Can I fixing cells after Annexin V staining?
Yes, cells can be fixed with formaldehyde after staining. Because Annexin V staining is dependent on calcium, all buffers used for washing and fixation should contain 1.25 mM CaCl2. Fixation may increase the background signal from Ethidium Homodimer III.
How is annexin V used in flow cytometry?
In flow cytometry, annexin V is commonly used to detect apoptotic cells by its ability to bind to phosphatidylserine, a marker of apoptosis when it is on the outer leaflet of the plasma membrane. Propidium Iodine: Propidium iodide is used as a DNA stain in flow cytometry to evaluate cell viability or DNA content in cell cycle analysis.
How to mix annexin V with dissolved Pi?
Add 1 μL of annexin V-FITC solution and 5 μL of dissolved PI to 100 μL of the cell suspensions prepared as given in step 1. Mix gently. Keep tubes on ice and incubate for 15 minutes in the dark. Add 400 μL of ice-cold 1X binding buffer and mix gently.
How is propidium iodide used in flow cytometry?
Propidium iodide is used as a DNA stain in flow cytometry to evaluate cell viability or DNA content in cell cycle analysis. Propidium Iodide cannot cross the membrane of live cells, making it useful to differentiate necrotic, apoptotic and healthy cells.
How is annexin V used to detect apoptosis?
However, in the intermediate stages of apoptosis, PS is translocated from the inner to the outer leaflet of the membrane, exposing PS to the external cellular environment where it can be detected. Highly fluorescent annexin V conjugates provide quick and reliable detection methods for studying the externalization of phosphatidylserine.
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