Experiment Principle:

Annexin V is a phospholipid binding protein, which can bind to cell membrane of early apoptotic cell, it is one of sensitive indicators for detection of early apoptotic cells. When cells undergo apoptosis, membrane phosphatidylserine will flip outside from inside of plasma membrane. Annexin V has high affinity to phosphatidylserine, thus it will bind to phosphatidylserine exposed in the cell outside. During apoptosis, eversion of phosphatidylserine occurs earlier than the alteration of nucleus; Hence, comparing with detection of DNA fragments, Annexin V can detect apoptotic cells in earlier time. Eversion of phosphatidylserine can also occur in cell necrosis, so Annexin V is often used with dye of nucleic acid(such as PI or 7-AAD) to distinguish the apoptotic cell (Annexin V+ / dye of nucleic acid-) and dead cell (Annexin V+ / dye of nucleic acid+). Through detecting signal strength of fluorescence-labeled Annexin V and nucleic acid dye, it can determine the state of cell apoptosis by flow cytometry.

Experiment steps:

1. Cells are collected (approximately 1×106 cells/sample), and centrifuged at 500~1000r/min for 5min, then discard culture medium.

2. Add 1 mL PBS buffer to re-suspended cells, cell suspension is centrifuged at 500~1000r/min centrifugal for 5min, then discard the PBS.

3. Add 1*Binding Buffer, re-suspended cells to prepare 1*106/mL cell suspension.

4.Take 100 μL cell suspension to a new tube, add Annexin V (5 μL), mix gently and incubate the mixture for 10 minutes at room temperature in the dark, then add the PI dye (5 μL), incubate them for 5 minutes at room temperature in the dark. Control and samples to be tested are set as follows:

5. Add PBS buffer to a final volume of 500 μL. Mix gently, and detect in the machine within 1 hour.

The result analysis, for example:

As figure, Annexin V+/PI-（fourth quadrant）are apoptosis cells，Annexin V+/PI+（first quadrant）are dead cells，Annexin V-/PI-（third quadrant）are normal cells.