How does Annexin V assay work?
The assay combines annexin V staining of PS and PE membrane events with the staining of DNA in the cell nucleus with propidium iodide (PI) or 7-Aminoactinomycin D (AAD-7), distinguishing viable cells from apoptotic cells and necrotic cells. Detection occurs by flow cytometry or a fluorescence microscope.
How does Annexin V 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 PI work?
The Annexin V corresponding signal provides a very sensitive method for detecting cellular apoptosis, while propidium iodide (PI) is used to detect necrotic or late apoptotic cells, characterized by the loss of the integrity of the plasma and nuclear membranes.
What does Annexin V specifically label?
Annexin V and propidium iodide (PI) labeling of cells is a technique used to identify cell death, and distinguish between its different pathways: apoptosis, or programmed cell death, and necrosis.
What does propidium iodide stain?
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.
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.
What does annexin V stain?
Annexin V, a 36-kDa calcium-binding protein, binds to PS; therefore, fluorescently labeled Annexin V can be used to detect PS that is exposed on the outside of apoptotic cells. Annexin V can also stain necrotic cells because these cells have ruptured membranes that permit Annexin V to access the entire plasma membrane.
How do you use propidium iodide in flow cytometry?
Resuspend cells in an appropriate volume of Flow Cytometry Staining Buffer. Add 5 µL of Propidium Iodide Staining Solution or 7-AAD Staining Solution per 100 µL of cells. Incubate for 5–15 minutes on ice or at room temperature. Do not wash cells.
What is annexin V and propidium iodide labeling?
Annexin V and propidium iodide (PI) labeling of cells is a technique used to identify cell death, and distinguish between its different pathways: apoptosis, or programmed cell death, and necrosis. Cells undergo distinct morphological changes depending on the pathway.
What is propidium iodide used for in cell culture?
Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability. The Annexin V/PI protocol is a commonly used approach for studying apoptotic cells.
What is the annexin V/Pi protocol?
The Annexin V/PI protocol is a commonly used approach for studying apoptotic cells. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells.
How can I induce apoptosis by using annexin V-FITC?
Alternatively, we would recommend this protocol: A. Incubation of cells with annexin V-FITC. 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.