Is annexin V specific for apoptosis?
Annexin V was shown to interact strongly and specifically with PS and can be used to detect apoptosis by targeting for the loss of plasma membrane asymmetry. Labeled annexin V can be applied both in flow cytometry and in light microscopy in both vital and fixed material by using appropriate protocols.
What is annexin V pi?
The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells3. 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 4,5.
What does annexin V measure?
Annexin V used for measuring apoptosis in the early events of cellular cytotoxicity. Cytometry.
What is Annexin V staining?
Annexin V staining is a common method for detecting apoptotic cells. Thermo Fisher Scientific offers high-quality fluorescent annexin V conjugates as standalone reagents and in a variety of kits for use in flow cytometry and for imaging suspension cells.
What happens to phosphatidylserine during apoptosis?
Phosphatidylserine exposure during apoptosis reflects bidirectional trafficking between plasma membrane and cytoplasm | Cell Death & Differentiation.
What is Pi in flow cytometry?
Propidium iodide (PI) is a membrane impermeant dye that is generally excluded from viable cells. It binds to double stranded DNA by intercalating between base pairs. PI is excited at 488 nm and, with a relatively large Stokes shift, emits at a maximum wavelength of 617 nm.
How does flow cytometry detect apoptosis?
One of the classical flow cytometric methods to detect apoptosis is using annexin V binding to phosphatidylserine residues normally located within the plasma membrane. Phosphotidylserine residues are externalised during apoptosis, so only cells that have decided to die will be detected by annexin V binding.
Can you fix annexin V?
In my experience, it is possible to fix annexin V stained cells and see great results on the flow. Stain cells with annexin V (in calcium-rich binding buffer). Wash cells in binding buffer to remove unbound AxV. Fix in your choice of fixative (diluted in binding buffer).
How do you calculate apoptosis?
The apoptotic index was calculated by dividing the total number of apoptotic bodies by the total number of intact carcinoma cells and multiplying by 100.
What is annexin V flow cytometry?
Annexin-V – Cell membrane changes In general, though not always, this is an early event in apoptosis and is thought to be a signal to neighbouring cells that a cell is ready to be phagocytosed. Annexin-V is a specific PS-binding protein that can be used to detect apoptotic cells.
What is the function of phosphatidylserine?
Phosphatidylserine (abbreviated Ptd-L-Ser or PS) is a phospholipid and is a component of the cell membrane. It plays a key role in cell cycle signaling, specifically in relation to apoptosis. It is a key pathway for viruses to enter cells via apoptotic mimicry.
Why does propidium iodide staining indicate apoptosis?
Propidium Iodide Propidium Iodide (PI) is a fluorescent DNA-binding dye. It freely penetrates cell membranes of dead or dying cells, but is excluded from viable cells. These properties favor the use of PI for evaluation of (apoptotic) cell death or for cell cycle analysis.
Can you fix 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.
Is Annexin V an antibody?
Anti-annexin V antibodies (aANVAs) are detected in various diseases like rheumatoid arthritis, systemic lupus erythematosus and anti-phospholipid antibody syndrome. The study of ANV in Acute Myocardial Infarction (AMI) might shed light on hypercoagulability mechanisms in the pathogenesis of acute coronary syndromes.
What is markers of apoptosis?
The markers most commonly used for in vitro detection of apoptosis include caspase-3/7 activity and phosphatidylserine (PS) exposure on the outer leaflet of the cell membrane.
What is apoptosis rate?
Apoptotic rate: a new indicator for the quantification of the incidence of apoptosis in cell cultures.
How does phosphatidylserine act as a marker of apoptosis?
The exposure of phosphatidylserine (PS) on the outer plasma membrane has long been considered a unique feature of apoptotic cells. Together with other “eat me” signals, it enables the recognition and phagocytosis of dying cells (efferocytosis), helping to explain the immunologically-silent nature of apoptosis.
What does propidium iodide detect?
Propidium iodide (PI) is a popular red-fluorescent nuclear and chromosome counterstain. Since propidium iodide is not permeant to live cells, it is also commonly used to detect dead cells in a population. PI binds to DNA by intercalating between the bases with little or no sequence preference.
What does propidium iodide stain?
Propidium iodide is a membrane-impermeable fluorescent DNA stain. It is used to stain apoptotic cells for flow cytometry, in situ hybridization, and immunohistochemistry applications Belloc et al (1994). It can also be used to differentiate between apoptotic and necrotic cell death.
What is the role of annexin V in late stage apoptosis?
However, in late-stage apoptosis, the cell membrane loses integrity, thereby allowing annexin V to also access PS in the interior of the cell. A viability dye can be used to resolve these late-stage apoptotic and necrotic cells (annexin V, viability dye-positive) from the early-stage apoptotic cells (annexin V positive, viability dye-negative).
What viability stains are available with annexin V apoptosis detection kits?
Current viability stains available with the eBioscience Annexin V Apoptosis Detection kits include FITC, eFluor 450, allophycocyanin (APC), phycoerythrin (PE), PE-cyanine7, and biotin. For Research Use Only. Not for use in diagnostic procedures.
How do you detect apoptosis in flow cytometry?
Easily distinguish cells undergoing early and late apoptosis with eBioscience and Annexin V Apoptosis Detection kits for flow cytometry analysis. These kits utilize viability dye-conjugated annexin V to label phosphatidylserine (PS), an early marker of apoptosis, on the extracellular membrane.
Can fixable viability dye eFluor 450 be used with annexin V apoptosis detection kits?
Note: Fixable Viability Dye eFluor 450 is not recommended for use with Annexin V Apoptosis Detection Kits. Annexins are a family of calcium-dependent phospholipid-binding proteins that preferentially bind phosphatidylserine (PS). Under normal physiologic conditions, PS is predominantly located in the inner leaflet of the plasma membrane.