Annexin V is a membrane protein which binds directly to phosphatidyl serine (PS) which is normally expressed internally, but upon apoptosis initiation moves to the cell surface. Annexin V is therefore a common tool for detecting early initiation of cell death.

Due to the calcium dependence of the Annexin V: PS interaction it is critical to avoid buffers containing EDTA or other calcium chelators during Annexin V : PS binding. Many mediums and buffers contain one or more of these incompatible reagents, therefore it is recommended to use a specific Annexin Binding buffer which is included in all Annexin kits but can also be purchased separately, cat# BMS500BB.

Customers often ask if it is possible to co-stain Annexin V with:

1) A viability dye &

2) Surface staining antibodies simultaneously in Annexin Binding buffer.

While PI and 7-AAD are compatible with the Annexin Binding Buffer, it is not recommended to stain with the Fixable Viability dyes (FVDs) simultaneously with Annexin V, since FVDs bind to any protein with a free amine group. Instead it is recommended using a serum/protein-free PBS buffer and staining with FVD before Annexin staining.

There is an optimized protocol for Annexin staining with FVD and surface/intracellular antibody staining which can be found in the Best Protocols section under Flow Cytometry (FACS) Protocols: Annexin V staining: