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 PI?
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 is Annexin V binding buffer?
The Annexin V Binding Buffer is a 10X concentrate composed of a 0.2 µm sterile filtered 0.1M Hepes (pH 7.4), 1.4M NaCl, and 25 mM CaCl2 solution. Prior to staining cells, an appropriate quantity of a 1X working solution should be made by diluting the 10X concentrate 1:10 with distilled water.
Where is annexin V located?
The human vascular anticoagulant annexin V is a 35–36 kDa, Ca2+-dependent phospholipid-binding protein that has a high affinity for the anionic phospholipid phosphatidylserine (PS). In normal healthy cells, PS is located on the cytoplasmic surface of the plasma membrane.
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.
How do you stain Annexin V?
Add 5 μL of fluorochrome-conjugated Annexin V to 100 μL of the cell suspension. Incubate 10-15 minutes at room temperature. Protect from light. Add 2 mL of 1X binding buffer and centrifuge at 400-600 x g for 5 minutes at room temperature.
Does PI stain apoptotic cells?
The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6.
What is the purpose of binding buffer?
The chaotropic salt binding buffer allows the highest DNA binding of any column method. Powerful wash buffers remove all traces of protein and salt. DNA is eluted in a low-salt buffer to allow for pH stabilization of the DNA in storage.
How does propidium iodide staining work?
Propidium iodide (or PI) is a fluorescent intercalating agent that can be used to stain cells and nucleic acids. PI binds to DNA by intercalating between the bases with little or no sequence preference. PI also binds to RNA, necessitating treatment with nucleases to distinguish between RNA and DNA staining.
How can you tell the difference between annexin V and Cat?
Both healthy cells and apoptotic cells possess phosphatidylserine on the cell surface, which can be detected with Annexin V, but apoptotic cells have significantly more of it. You can easily tell the difference between these two populations with flow cytometry, because flow cytometers are more sensitive and have a higher throughput.
What is the difference between FITC annexin V and Pi?
For example, cells that are considered viable are both FITC Annexin V and PI negative while cells that are in early apoptosis are FITC Annexin V positive and PI negative, while cells that are in late apoptosis or already dead are both FITC Annexin V and PI positive.
How does annexin V stain in early apoptosis?
These cells will stain with Annexin V but not with viability dyes, thus distinguishing cells in early apoptosis. In late stage apoptosis, the cell membrane loses integrity, thereby allowing Annexin V to also access PS in the interior of the cell.
What kind of staining is used for annexin V?
Once on the outer surface of the membrane, PS can be detected by fluorescently labeled Annexin V in a calcium-dependent manner. In early-stage apoptosis, the plasma membrane excludes viability dyes such as propidium iodide (PI), 7-AAD, as well as Invitrogen™ Fixable Viability Dyes (FVD) such as eFluor™ 660, eFluor™ 506 or eFluor™ 780.
