Annexin V Apoptosis Kit [FITC]

Annexin V Apoptosis Kit [FITC] can identify apoptosis at an earlier stage than kits based on DNA fragmentation in the nucleus. However, it is like most assays and has 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 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 because the dead cells will stain with both Annexin V-FITC and PI (see image). 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.