Apoptosis and Necrosis

Apoptosis and Necrosis using Flow Cytometry

Using Flow Cytometry an investigator can study a cell population and see what percentage are undergoing apoptosis or have already died. A cell population is treated for a set amount of time and then fixed. Once fixed the population is stained using Propidium Iodide (PI) which stains dead cells and a FITC labeled antibody (Annexin V) to phosphatidylserine (PS). PS is a phospholipid normally found inside the cell membrane, but which is transferred to the outer leaflet of the cell membrane and is used as an indicator of apoptosis. The resulting flow data looks like this:

Apoptosis results data

From this experiment we can see that 29.8% of the population is apoptotic, with 2.1% of the population being already dead or with a compromised membrane.

Another experiment which can be performed is the TUNEL assay. This assay measures DNA fragmentation using an enzyme which incorporates biotinylated uridine. A FITC labeled antibody against biotin then binds the uridine. When run through the flow cytometer the FITC fluoresces. You can use this assay to see how many of the cells have fragmented DNA, another indication of apoptosis.

Additional Information:

• BD FACS Canto Flow Cytometer page

• Flow Cytometer Instructions