What does Annexin V positive mean?
What does Annexin V positive mean?
Annexin V positive – PI positive staining indicate cells are in necrosis (post-apoptotic necrosis or late apoptosis).
What does Annexin V stain?
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).
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 is used in conjunction with labelled Annexin V.
What is the purpose of propidium iodide in this assay?
Propidium iodide is used as a DNA stain in flow cytometry to evaluate cell viability or DNA content in cell cycle analysis, or in microscopy to visualize the nucleus and other DNA-containing organelles.
Where is annexin V located?
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.
Can I fix annexin V stained cells?
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.
Why do you use annexin V and propidium iodide?
Staining with annexin V and propidium iodide (PI) provides researchers with a way to identify different types of cell death—either necrosis or apoptosis. This technique relies on two components.
What is the protocol for annexin V staining?
Alternatively, we would recommend this protocol: A. Induce apoptosis by desired method. Collect 1–5 x 10 5 cells by centrifugation. Resuspend cells in 500 µL of 1X binding buffer. Add 5 µL of annexin V-FITC and 5 µL of propidium iodide (PI, optional). Incubate at room temperature for 5 min in the dark.
Can a cell be both Pi and annexin stained?
This DNA binding dye molecule is again membrane impermeable, and can only enter a cell when the membrane is ruptured. Therefore, if a cell is only annexin stained it is early apoptotic, whereas a cell that is both PI and annexin stained could be either necrotic or late apoptotic.
How long to incubate annexin V for staining?
Add 5 µL of annexin V-FITC and 5 µL of propidium iodide (PI, optional). Incubate at room temperature for 5 min in the dark. Proceed to B or C below depending on the analysis method.