Thursday, December 6, 2012

Applications Thursday: Simple Apoptosis Assay in an Absorbance Microplate Reader

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. Apoptosis is a common pathway that can go wrong in many cancers and it is a highly researched area for different cell types. Common methods for apoptosis analysis include:
  • Caspase activity assay, either colorimetric, fluorescent, luminescent or antibody based
  • TUNEL assay, based on DNA fragmentation.
  • Annexin-V assay, based on the binding of dye or fluorescently-conjugated Annexin-V to phosphatidyl serine which has translocated to the cell membrane exterior during apoptosis.
A simple and easy-to-use apoptosis assay, Bicolor's APOPercentage Apoptosis Assay™, was performed on BMG LABTECH's SPECTROstar Nano microplate reader. This assay format takes advantage of the phosphatidyl serine (PS) that is found on the exterior cell surface when apoptosis begins to occur. The APOPercentage dye, which binds to PS, is then taken up by cells undergoing apoptosis. The dye can be quantified by an absorbance instrument such as the SPECTROstar Nano

Table 2
Colorimetric Quantification. Graph Showing Effect of Hydrogen Peroxide (0 – 10mM) on CHO Cells. Results expressed as mean absorbance for triplicate wells ± S.E.M. (n = 3). Exposure time to H2O2 was 4 hours.