Annexin V is a phospholipid binding protein with high affinity for binding to phosphotidyl serine (PS), which is a phospholipid present exclusively in the inner leaflet of the cell membrane.
Annexin V does not bind to normal cells because of its inability to penetrate the lipid bilayer.
During apoptosis, due to changes in membrane permeability, PS is translocated to the outer surface of the membrane with which Annexin V binds with high affinity in the presence of Ca .
Detection of cell surface PS with Annexin V thus serves as a marker for apoptotic cells. Similarly, propidium iodide (PI), which specifically stains the dead cells, is used as a marker for necrosis.
Adopted from from BioRender
Apoptosis Principle Assay
In this assay, we are using fluorescently tagged Annexin V and PI as markers for apoptotic and necrotic cells respectively.
The number of annexin positive cells and PI positive cells are counted under the fluorescence microscope to estimate the extent of apoptosis and necrosis in response to specific drug treatment.
Materials Required for Apoptosis Assays
1. Phosphate Buffer Saline (PBS) (i.e. 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4.2H2O and 2 mM KH2PO4, pH 7.4).
2. Annexin binding buffer (10mM HEPES (pH 7.5), 140mM NaCl, 2.5mM CaCl2)
3. Annexin V FITC (1mg/ml in PBS)
4. Propidium iodide (1mg/ml in PBS)
5. Fluorescence microscope with green and blue filters
Apoptosis Assay Procedure
1. Remove the medium and wash the cells with PBS thrice and once with annexin binding buffer
2. Add 100μl of annexin binding buffer and subsequently add annexin V-FITC (final concentration should be between 2 μg/ml)
3. Incubate the cells in dark at 37oC for 30 minutes
4. Add PI (Final concentration 1 μg/ml) and keep at RT for 10 minutes
5. Wash the cells twice with annexin binding buffer and observe under the fluorescence microscope
6. Capture atleast 5 fields for each treatment and don’t forget to take phase contrast images of all the fields.
Apoptosis Assay Calculation
Percentage of apoptotic cells = [no. of annexin positive cells/total no. of cells] x 100 Percentage of necrotic cells = [no. of PI positive cells/total no. of cells] x 100