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FITC Annexin V is used to quantitatively determine the percentage of cells within a population that are actively undergoing apoptosis. It relies on
the property of cells to lose membrane asymmetry in the early phases of apoptosis. In apoptotic cells, the membrane phospholipid
phosphatidylserine (PS) is translocated from the inner leaflet of the plasma membrane to the outer leaflet, thereby exposing PS to the external
environment. Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for PS, and is useful for identifying
apoptotic cells with exposed PS. Propidium Iodide (PI) is a standard flow cytometric viability probe and is used to distinguish viable from
nonviable cells. Viable cells with intact membranes exclude PI, whereas the membranes of dead and damaged cells are permeable to PI. Cells that
stain positive for FITC Annexin V and negative for PI are undergoing apoptosis. Cells that stain positive for both FITC Annexin V and PI are
either in the end stage of apoptosis, are undergoing necrosis, or are already dead. Cells that stain negative for both FITC Annexin V and PI are
alive and not undergoing measurable apoptosis.
Reagents
1. FITC Annexin V: Included. Use 5 μl per test.
2. Propidium Iodide (PI): Not Included
. PI (cat.no. 556463) is a convenient, ready-to-use nucleic acid dye. Use up to 10 μl per test of a 50 μg/ml
solution.
3. 10× Binding Buffer: Not Included. 0.1 M Hepes (pH 7.4), 1.4 M NaCl, 25 mM CaCl2. Store at 4°C. Alternatively, catalog number 556454
may be purchased.
556419 Rev. 2 Page 3 of 4
Staining
1. Wash cells twice with cold PBS and then resuspend cells in 1× Binding Buffer at a concentration of 1 × 10e6 cells/ml.
2. Transfer 100 μl of the solution (1 × 10e5 cells) to a 5 ml culture tube.
3. Add 5 μl of FITC Annexin V.
4. Add 10 μl PI. The optimal concentration of PI may vary among cell lines where 10 μl of a 50 μg/ml stock is most likely the maximum to be
required. Less may yield optimal results in some experimental systems.
5. Gently vortex the cells and incubate for 15 min at RT (25 °C) in the dark.
6. Add 400 μl of 1× Binding Buffer to each tube. Analyze by flow cytometry within 1 hr.
SUGGESTED CONTROLS FOR SETTING UP FLOW CYTOMETRY
The following controls are used to set up compensation and quadrants:
1. Unstained cells.
2. Cells stained with FITC Annexin V (no PI).
3. Cells stained with PI (no FITC Annexin V).
Other Staining Controls:
A cell line that can be easily induced to undergo apoptosis should be used to obtain positive control staining with FITC Annexin V and/or FITC
Annexin V and PI. It is important to note that the basal level of apoptosis and necrosis varies considerably within a population. Thus, even in the
absence of induced apoptosis, most cell populations will contain a minor percentage of cells that are positive for apoptosis (FITC Annexin V
positive, PI negative or FITC Annexin V positive, PI positive).
The untreated population is used to define the basal level of apoptotic and dead cells. The percentage of cells that have been induced to undergo
apoptosis is then determined by subtracting the percentage of apoptotic cells in the untreated population from percentage of apoptotic cells in the
treated population. Since cell death is the eventual outcome of cells undergoing apoptosis, cells in the late stages of apoptosis will have a damaged
membrane and stain positive for PI as well as for FITC Annexin V. Thus the assay does not distinguish between cells that have already undergone
an apoptotic cell death and those that have died as a result of necrotic pathway, because in either case the dead cells will stain with both FITC
Annexin V and PI.
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