Detection Principle

The distribution of phospholipids in normal cell membranes is asymmetric. phosphatidylserine (PS) is located on the inner surface of the cell membrane in living cells, and during apoptosis, the cell membrane undergoes a change, and PS is flipped from the inner surface of the cell membrane to the outer surface of the cell membrane. Annexin V specifically recognizes PS on the surface of apoptotic cells, whereas PS from living cells is located on the inner surface of the cell membrane and is unable to bind specifically to Annexin V.

The PS of necrotic cells also flips from the inner surface of the cell membrane to the outer surface of the cell membrane, and Annexin V also recognizes the PS on the surface of necrotic cells, so Annexin V is unable to distinguish between necrotic and apoptotic cells. In contrast, PI dye was able to bind to intracellular DNA and distinguish between necrotic cells and living cells. The cell membrane of early apoptotic cells and living cells is still intact, and PI dye cannot freely pass through the cell membrane to enter the cell and bind to the DNA, so PI dye cannot label apoptotic cells and living cells, while PI dye is able to pass through the cell membrane of the necrotic cells and bind to the DNA inside the cell, and the PI dye in the necrotic cells will emit red fluorescence after being excited by the 488 nm laser, which will be received by the corresponding channel. So Annexin V and PI can be used simultaneously to distinguish between living cells, early apoptotic cells and late apoptotic or necrotic cells.

Fig. 2 Principle of Annexin V / PI detection

Flow Cytometry Detection

The maximum excitation wavelength of FITC is 488 nm, the maximum emission wavelength is 525 nm, and the green fluorescence of FITC is detected in the FL1 channel. The maximum excitation wavelength of PI-DNA complex is 535 nm, the maximum emission wavelength is 615 nm, and the red fluorescence of PI is detected in the FL2 or FL3 channel. The analysis was performed with software such as CellQuest, and a two-color scatter plot was drawn with FITC as the horizontal coordinate and PI as the vertical coordinate. In typical experiments, the cells can be divided into three subpopulations, with live cells having only very low-intensity background fluorescence, early apoptotic cells having only strong green fluorescence, and late apoptotic cells having double staining with green and red fluorescence.

Fig.3 Flow Cytometry Detection

Frequently Asked Questions

Q: Can the Annexin V apoptosis assay be applied to plants or bacteria (prokaryotes)?

A: Yes, but it is necessary to prepare protoplasts because plant cells or bacteria (prokaryotes) contain cell walls. The specific dosage of staining solution only needs to submerge the cells, and the staining time is somewhat different for different cells.

Q: How to judge the result of the experiment?

A: Live cells (Annexin V-/PI-), early apoptotic cells (Annexin V+/PI-), late apoptotic cells and necrotic cells show double positivity (Annexin V+/PI+), naked nuclei (Annexin V-/PI+).

Q: What is the difference between Annexin V and TUNEL?

A: Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) is a staining method used to identify intracellular DNA fragmentation sites - a hallmark feature of late apoptosis.

Annexin V staining recognizes the early stages of apoptosis by binding to PS residues exposed outside the cell due to loss of cell membrane asymmetry.

Q: Is it possible to perform the assay using only Annexin V-FITC without detecting PI?

A: Yes, it is possible. For special test samples, such as red blood cells without nuclei, or the use of the drug Doxorubicin (Adriamycin) which interferes with PI detection, it is possible to perform the assay using only Annexin V-FITC in both cases.

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