By Can you fix cells for 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.
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-FITC work?
Annexin V is a 35-36 kDa Ca2+ dependent phospholipid-binding protein that has a high affinity for PS, and binds to cells with exposed PS. While cells that are in early apoptosis are Annexin V-FITC positive and PI negative and cells that are in late apoptosis or already dead are both FITC Annexin V and PI positive.
What does annexin V positive mean?
> Annexin V positive – PI positive staining indicate cells are in necrosis (post-apoptotic necrosis or late apoptosis).
Does DAPI stain live or dead cells?
DAPI is a fluorescent stain that binds strongly to A-T-rich regions in DNA. DAPI and PI only inefficiently pass through an intact cell membrane and, therefore, preferentially stain dead cells. TMRM is a cell-permeable fluorescent dye that is sequestered to active mitochondria, and hence labels live cells.
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.
What color is annexin V?
Cells that have bound annexin V-FITC will show green staining in the plasma membrane. Cells that have lost membrane integrity will show red staining (PI) throughout the nucleus and a halo of green staining (FITC) on the cell surface (plasma membrane).
What kind of staining does annexin V show?
Cells that have bound annexin V-FITC will show green staining in the plasma membrane. Cells that have lost membrane integrity will show red staining (PI) throughout the nucleus and a halo of green staining (FITC) on the cell surface (plasma membrane). . The above is an example protocol for PS exposure detection using annexin V,
How to stain annexin V for flow cytometry?
Resuspend cells in 1X Binding Buffer at 1-5 x 10 6 cells/mL. 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 1X binding buffer and centrifuge at 400-600 x g for 5 minutes at room temperature.
How is annexin V used to identify dead cells?
Cells were then treated with Annexin V, Alexa Fluor 488 conjugate to identify apoptotic cells and with propidium iodide to identify dead cells, followed by flow cytometric analysis.
Where is annexin V located on the cell membrane?
The human vascular anticoagulant annexin V is a 35–36 kDa, Ca 2 +-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 cell membrane.