Phosphatidylserine Externalization in Apoptosis
In healthy cells, phosphatidylserine (PS), an anionic phospholipid, is actively translocated to the inner leaflet of the cell membrane. During apoptosis, this distribution is randomized, resulting in the appearance of PS on the outer leaflet. Exposed PS can be detected by the calcium dependent binding of Annexin V. The anticoagulant protein, Annexin V, preferentially binds PS with a stoichiometry of ~50 PS monomers per protein molecule. When conjugated to a reporter molecule (FITC, Cy5, PE, EGFP, etc.), bound Annexin V can be detected by flow cytometry or by microscopy.
pSIVA-IANBD is an advanced annexin-based probe that addresses the key limitation of traditional PS detection assays, background fluorescence.
Browse our tools to detect exposed PS including:
Principles of the Annexin V Apoptosis Assay and Dye Exclusion Method
Note: Due to the permeability of necrotic cells, labeled Annexin V may be able to penetrate the outer membrane and complex with PS in the inner leaflet. In this case, a positive signal of PS translocation in necrotic cells is an artifact. If false positive results from necrosis are of concern, consider including Necrostatin-1, a specific inhibitor of necrosis, in experiments.
Detection of Apoptotic Dexamethasone-treated Thymocytes by Annexin V Staining. Thymocytes were left untreated (A) or treated with 100 nM dexamethasone for 15.5 hours (B), and then stained using Annexin V-FITC and PI provided in the Annexin V-FITC Apoptosis Detection Kit [NB4830-01-K]. Analysis courtesy of Dr. C.M. Knudson, Howard Hughes Medical Institute, St. Louis, MO.
Analysis Tip: When combined with the dye exclusion method, the Annexin V assay enables further discrimination of cells undergoing apoptosis.
| Cell Population | Staining |
|---|---|
| Viable | Annexin V negative – PI negative |
| Early Apoptosis | Annexin V positive – PI negative |
| Late Apoptosis / Post-apoptotic Necrosis | Annexin V positive – PI positive |