GFP-Certified™ Apoptosis/Necrosis Detection Kit is a multiplex assay that distinguishes between healthy, early apoptotic, late apoptotic and necrotic cells, compatible with GFP and other green fluorescent probes.

The transition from apoptosis to necrosis is a loosely defined continuum that necessitates recognition of the various stages of the process. The display of phosphatidylserine (PS) on the extracellular face of the plasma membrane remains a unifying hallmark of early apoptosis. Phospholipid-binding proteins such as Annexin V bind with a high affinity to PS, in the presence of Ca2+. Given that Annexin V is not cell permeable, the binding of externalized PS is selective for early apoptotic cells. Similarly, loss of plasma membrane integrity, as demonstrated by the ability of a membrane-impermeable DNA intercalating dye to label the nucleus, represents a straightforward approach to demonstrate late stage apoptosis and necrosis. Considering that Annexin V is commonly conjugated to fluorescein (a.k.a. FITC), apoptosis detection in green fluorescent protein (GFP)-expressing cells can be problematic.

The spectral overlap between the emission profiles of these two fluorophores makes differentiation between signals difficult or impossible, thus compromising data quality overall. While useful for analysis of apoptosis and necrosis in cells that do not express any fluorescent proteins, the GFP-Certified™ Apoptosis/Necrosis Detection System was specifically designed for use with GFP-expressing cell lines and cells expressing blue or cyan fluorescent proteins (BFPs, CFPs).

Additionally, the kit is suitable for use with live or post-fixed cells in conjunction with probes, such as labeled antibodies, or other fluorescent conjugates displaying similar spectral properties as fluorescein or coumarin. This kit includes all the necessary reagents for determination of early and late stages of apoptosis as well as necrosis. An Annexin V-EnzoGold (enhanced Cyanine-3) conjugate enables detection of apoptosis distinct from fluorescein or GFP.

The Necrosis Detection Reagent (Red) similar to the red-emitting dye 7-AAD, facilitates late apoptosis and necrosis detection. This kit also includes an Apoptosis Inducer (Staurosporine) for use as a positive control. Detection reagents can be used separately or in combination for either single or multiplexed applications respectively. This kit also provides quick and accurate results via flow cytometry and fluorescence/confocal microscopy.