The Biocolor Cell-APOPercentage™ Apoptosis Assay is a detection and MEASUREMENT system which allows the user to monitor the incidence of apoptosis (programmed cell death) in mammalian, anchorage dependent cells during in vitro culture.
The assay uses a dye that is selectively imported by cells undergoing apoptosis. Necrotic cells cannot retain the dye and therefore are not stained.
Two different protocols are available. The approach adopted will depend on the user’s choice of quantification method:
- Colorimetric protocol - The dye that accumulates in 30 minutes within labeled cells is released into solution and the concentration of released intracellular dye measured using a microplate colorimeter.
- Analytical Digital Photomicroscopy protocol - The dye used has a purple-red color, which allows detection of apoptosis with a conventional inverted microscope. Photomicrograph images obtained are transferred to a computer where the stained area is counted in pixels using downloadable software (ImageJ).
The manual describes the colorimetric protocol in a 24-well plate format and the Analytical Digital Photomicroscopy protocol in a 96 well plate. Alternative microwell plates, microscope chamber slides and T-flasks are also suitable for use with the assay.
Videos on how the Apoptosis Assay Works
A single apoptotic cell within a microwell cell population.
Assay Run Time
What type of Cells can be used
Established cell lines are suitable. Cells should be anchorage-dependent. In order to enhance attachment cells exposed to toxic agents may be grown in gelatin.
Diagram Showing The Asymmetric Phospholipid Composition of a Viable and an Apoptotic Mammalian Cell Membrane
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 Cell-Apopercentage dye binds to the externally exposed phosphatidylserine and the dye is taken up into the cell via this phosphatidylserine transmembrane movement.
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 Cell Apopercentage APOPTOSIS Assay can be used on its own or alongside other apoptosis detection assays, such as a caspase activity assay, a tunnel 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).
In the links below you will find the Cell-APOPercentage™ Apoptosis Assay protocols, publications that reference the Cell-APOPercentage™ Apoptosis Assay, and the Cell-APOPercentage™ Apoptosis Assay MSDS.
- Product Data Sheet (PDS) with Colorimetric Protocol
- Product Data Sheet (PDS) with Analytical Digital Photomicroscopy Protocol
- References to Cell-APOPercentage™ Apoptosis Assay (2019)
- Material Safety Data Sheet (MSDS)
Ilex Life Sciences LLC is an official distributor of Biocolor products.