Description
The Biocolor Cell-APOPercentage™ Apoptosis Assay is a detection and measurement system which allows the user to monitor the occurrence of apoptosis in mammalian, anchorage-dependent cells during in vitro culture.
The assay uses a dye that is selectively imported by cells that are undergoing apoptosis. Necrotic cells cannot retain the dye and therefore are not stained.
| Limit of Detection | A single cell (via image analysis method) |
| Detection Method | Colorimetric (550 nm) or Image Analysis based |
| Measurements per kit | Sufficient for 4x24 well plates or 6x96 well plates |
| Suitable Samples | Adherent mammalian cells (in-vitro) |
| Assay Run Time | 1 hour |
Assay Principle
The mammalian cell membrane has been described as a semi‐fluid mosaic structure, composed of phospholipids with a diverse group of inserted proteins and some cholesterol. The phospholipids are the major components of the membrane and are arranged in the form of a ‘bi‐layer’; which is asymmetric in composition, structure, and function.
To ensure normal transmembrane functions the phospholipids must be maintained in an asymmetric composition. The process is regulated by ‘flippases’, which catalyse the active transport of aminophospholipids from the outer to inner monolayer. However, in cells undergoing apoptosis, flippase is overwhelmed by the action of another enzyme, termed ‘floppase’ or ‘scramblase’. The net effect is a scrambling of the phospholipid distribution between the inner and outer monolayers.
Diagram Showing The Asymmetric Phospholipid Composition of a Viable and an Apoptotic Mammalian Cell Membrane
The APOPercentage assay utilizes an intense, pink-colored dye reagent which is taken up during in-vitro culture by apoptosis-committed cells. This uptake occurs at the stage of Phosphatidylserine transmembrane movement, as produced by the flipflop mechanism. Dye uptake continues until blebbing occurs. No further dye can then enter the now defunct cell and the dye that has accumulated within the cell is not released (unlike necrotic cells which release dye).
Since the dye reagent is excluded or not retained by healthy or necrotic cells it therefore acts as a specific label for apoptotic cells.
Quantification Method
Labelled apoptosis cells may then be conveniently analyzed by the following methods:
Direct Analysis: The intense pink color of the labelled cells can be visually assessed using brightfield microscopy. Apoptosis in substrate-adherent cell populations is therefore readily quantified using image analysis techniques. Photomicrograph images obtained are transferred to a computer where the stained area is counted in pixels using downloadable software (ImageJ). This technique is the most sensitive with the ability of detecting one single apoptotic cell per well.
Colorimetry Protocol: Dye that accumulates within apoptotic cells is released into solution via addition of Dye Release Reagent. The concentration of this intracellular dye is then measured at 550nm using a microplate colorimeter/spectrophotometer.
The assay manual describes the colorimetric protocol in a 24-well plate format. Alternative microwell plates, microscope chamber slides and T-flasks are also suitable for use with the assay.
The APOPercentage assay kit does NOT require the use of a Flow Cytometer.
Videos on how the Apoptosis Assay Works
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.
Kit Contents
The APOPercentage™ Apoptosis Assay kit contains:
- APOPercentage Dye (1x 5 ml)
- Dye Release Reagent (1x 150 ml)
- Phosphate Buffered Saline (PBS) (1x 120 ml)
- 24-well starter plate.
- Assay kit manual.
The Colorimetric Protocol requires a Microplate Colorimeter / Spectrophotometer.
Additional 96-well plates will be required for use when reading dye absorbance values.
The Direct Detection Protocol requires an inverted stage microscope with an attached digital camera.
NOTE: Additional reagents (typically culture medium and suitable apoptosis treatments) may be required for sample preparation prior to assay. Consult manual or contact us for further details.
Application note
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).
Resources
Ilex Life Sciences LLC is an official distributor of Biocolor products.