Q. What is the sensitivity of the Cell-APOPercentage assay?
A. A single apoptotic cell within a microwell cell population.
Q. How long does the Cell-APOPercentage assay take to run?
A. 1 hour.
Q. What types of cell can the assay be used with?
A. 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.
Q. What absorbance values can samples be measured at?
A. 550nm on a microplate colorimeter.
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 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).
Cell Attachment Using Gelatin
The addition of experimental apoptotic agents to the culture medium can often have an unfavourable effect on cell adhesion, resulting in detachment and ‘rounding-up’. Firm anchorage attachment of the cells, prior to adding the apoptotic test agent, can help to minimize this adverse effect. Extensive trials, using coated wells (collagen, gelatin, polylysine) found that a ‘deep layer’ of a weak gel of gelatin was particularly effective in providing uniform cell growth and preventing some cell loss caused by apoptotic inducers. Other cell types may differ and test trials to ascertain suitable matrix composition is recommended. If unacceptable cell loss in encountered with your test agent(s) consider growing the cells in gelatin.
Recommended Protocol for Cell Adhesion using Gelatin (volumes given for 24 well plate)
- Add 250μl of a 0.4% w/v gelatin solution in water to a 24-well plate.
- Seed cells at required density in 500μl culture medium on top of the gelatin. (The seeding density required to provide a confluent cell layer will be higher in presence of gelatin than in its absence).
- Incubate the plate at 37°C/5% CO2 until confluence is reached.
- Remove the gelatin and culture medium solution. Rinse cells once with 500μl of fresh medium.
- Carry out the APOPercentage Assay as detailed on inside cover of manual from step 2 onwards.