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APOPercentage™ Apoptosis Assay –
Apoptosis Detection
APOPercentage is a dye based assay suitable for mammalian cells that have a conventional phospholipid bilayer cell membrane. Phosphatidylserine transfer to the outside of the membrane during apoptosis permits the transport of the APOPercentage dye into the cell. APOPercentage Dye labelled cells can be viewed and quantified. Necrotic cells cannot retain the dye.
Assay sensitivity; a single apoptotic cell Assay run time; 1 hour
APOPTOSIS DETECTION
Apoptosis is a multistage process during which activity of caspase enzymes fluctuates, DNA becomes fragmented and phosphatidyl serine is transferred to the outside of the cell membrane. The Apopercentage dye binds to the externally exposed phosphatidylserine and the dye is taken up into the cell via this phosphatidylserine transmembrane movement.
The Asymmetric Phospholipid Composition of a Viable and an Apoptotic Mammalian Cell
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The choline phospholipids make up most of the lipid in the extracellular membrane. This organisational composition is essential for the normal functions of a viable cell, including the insertion of protein receptors and transporters between the phospholipid molecules. Maintaining the asymmetric composition is an energy dependant process involving the activity of enzymes, termed ‘flippases’. In apoptotic committed cells flippase regulation is either overwhelmed, or is inactivated by the activity of the enzyme ‘scramblase’ (floppase). The transfer of phosphatidylserine to the outside of the membrane permits the transport of the APOPercentage dye into the cell. The uptake of the dye is uni-directional, leading to dye accumulation within the cell. As the cell shrinks in volume, during the apoptotic process, the cell dye content becomes more concentrated. |
Apoptosis assays are widely available, each based on a different stage of the apoptotic process. In some cases apoptosis can be induced without detectable DNA degradation and caspase activity may be reduced at late stage apoptosis. Therefore, running two different apoptosis detection methods simultaneously is recommended to confirm that cell death is occurring due to apoptosis. The Apopercentage APOPTOSIS Assay can be used on its own or alongside other apoptosis detection assays, such as a caspase activity assay, a tunel assay (based on DNA fragmentation) or an annexin-V assay (based on binding of Annexin V to phosphatidyl serine that has translocated to the outside of the cell membrane during apoptosis).
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