Annexin V is a calcium-dependent phospholipid binding protein with high affinity for phosphatidylserine (PS), a membrane component normally localized to the internal face of the cell membrane. Early in the apoptotic pathway, molecules of PS are translocated to the outer surface of the cell membrane where Annexin V can readily bind them. The Guava Nexin® assay incorporates this PS-Annexin V binding as an indicator of early stage apoptotic cells.
The Guava Nexin® assay kit utilizes Annexin V-PE to detect PS on the external membrane of apoptotic cells. The cell impermeant dye 7-AAD is included in the Nexin kit as an indicator of membrane structural integrity. 7-AAD is excluded from live, healthy cells and early apoptotic cells, but permeates late stage apoptotic and dead cells. The following populations of cells can be distinguished in this assay:
Non-apoptotic cells: Annexin V(-) and 7-AAD(-)
Early apoptotic cells: Annexin V (+) and 7-AAD(-)
Late stage apoptotic and dead cells: Annexin V(+) and 7-AAD(+)