Annexin V assay

Annexin Cells

Adherent RAW cells treated with actinomycin-D, detached with Accutase, our cell detachment solution and stained with the PFS V-Annexin-V Kit

Apoptosis is the term that describes programmed cell death. It is believed to take place in the majority of animal and plant cells. It is a distinct event that triggers characteristic morphological and biological changes in the cellular life cycle. One of the early events that happens in the apoptotic pathway is the loss of plasma membrane asymmetry. In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular environment. Annexin V when labeled with a fluorescent tag, such as FITC, can be used with flow cytometry to measure this event. Annexin V is a 35-36 kDa Ca2+ dependent phospholipid-binding protein that has a high affinity for PS, and binds to cells with exposed PS. Annexin V- FITC staining can identify apoptosis at an earlier stage than our APO-BrdU or APO-Direct kits based on DNA fragmentation in the nucleus. However, it is like most assays and has it limitations. Since Annexin V staining precedes the loss of membrane integrity which accompanies the later stages of cell death resulting from either apoptotic or necrotic processes, staining with Annexin V-FITC is typically used in conjunction with a live/dead dye such as propidium iodide (PI) or 7-Amino-Actinomycin (7-AAD) to allow the investigator to identify early apoptotic cells (PI negative, Annexin V-FITC positive) from dead cells (PI positive, AnnexinV-FITC positive). Viable cells with intact membranes exclude PI, whereas the membranes of dead and damaged cells are permeable to PI. For this reason, Annexin V staining has to be performed on live cells as opposed to the APO-BrdU and APO-Direct assays which require para- formaldehyde/ethanol fixed cells.

The Annexin V assay works in the following manner: Cells that are viable are both Annexin V-FITC and PI negative. While cells that are in early apoptosis are Annexin V-FITC positive and PI negative and cells that are in late apoptosis or already dead are both FITC Annexin V and PI positive. This assay does not distinguish between cells that have undergone apoptotic death versus those that have died as a result of a necrotic pathway which the APO-BrdU assay can distinguish because in either case, the dead cells will stain with both Annexin V-FITC and PI. This is illustrated in the picture above. However, when apoptosis is measured over time, cells can be often tracked from Annexin V- FITC and PI negative (viable, or no measurable apoptosis), to Annexin V-FITC positive and PI negative (early apoptosis,membrane integrity is present) and finally to Annexin V- FITC and PI positive (end stage apoptosis and death). The movement of cells through these three stages suggests apoptosis. In contrast, a single observation indicating that cells are both Annexin V-FITC and PI positive, in of itself, reveals less information about the process by which the cells underwent their demise. For this reason, it is a good idea to analyze samples from multiple time points.

One of the important issues of doing this assay on the flow cytometer is correct instrument setup. The FITC signal from the Annexin V-FITC will bleed into the Propidium Iodide signal. For this reason, the fluorescent compensation on the flow cytometer must be correctly adjusted. PFS has included a solution to this issue in their Annexin V kit. These are fluorescent compensation controls. They are run before the experimental samples are analyzed to set up the flow cytometer. We have illustrated how the compensation controls should look below if the flow cytometer is set up correctly. The setup for the experiment is fairly trivial if controls of this sort are used..

We invite you to read the staining protocol of our Annexin V kit. It can be found on the upper right corner of this web page. Our Annexin V kit contains enough reagents to process 100 samples and 10 aliquots of each of the control cells. If you have any questions, do not hesitate to ask. Questions?

Reagents

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