Single tube 3-parameter apoptosis assay

A simple, yet improved (3-parameter, 3-colour) apoptosis assay 

The combination of Annexin V binding to detect phosphatidyl serine displayed on inverted plasma membranes of early apoptotic cells and a DNA-binding viability dye that permeates leaky plasma membranes denoting very late apoptotic/dead cells is a widely used simple, low-cost, single tube assay for cellular apoptosis.  

However, it is clear that these two plasma membrane "events" give little information about the actual events and progress of apoptosis and it would be therefore beneficial to include a further parameter that is earlier, independent, sentinel or pivotal for apoptosis or more correctly, programmed cell death.  

One such parameter might be the collapse of mitochondrial membrane potential (ΔΨm) observed proportionally with the reduction of binding of a commonly used rhodamine-based dye: TMRE (or TMRM).  

Historically, the most commonly used combination of Annexin V and viability dye has been Annexin-V-FITC and propidium iodide.  Unfortunately, the emission spectra of propidium iodide and TMRE/TMRM heavily overlap making this combination impractical.  

However, one can simply replace propidium iodide with the far-red fluorescing DNA-binding viability dye DRAQ7, which then permits combination of viability dye with TMRE and Annexin V enables the three parameters to be measured in a single-tube experiment.

An example of this, shown below, has been kindly provided by Dr. Lucia Piñon P Giraldez, Head of Advanced Light Imaging and Flow Cytometry Facilities, MRC Toxicology Unit, Cambridge, UK.  Annexin V is detected in the "FITC" channel, TMRE in "PE" and DRAQ7 in "APC-Cy7".

One might also consider caspase 3/7 activation reporters. These have, similarly, been combined with TMRM and DRAQ7 and demonstrated in time-lapse image-based assays.

DRAQ7 offers new assay design alongside consistent product quality and ease-of-use backed by 1700+ citations (as of Nov. '23) in peer-reviewed journal articles.

WHERE TO BUY

DRAQ7™ offers a significant advantage in that it is truly cross-platform compatible, able to be the cell death reporter in flow cytometry, plate-based cytometers (Sartorius CeligoS), imaging flow cytometry (e.g. Cytek® Amnis® Imagestream®, real-time imaging analysis (e.g. on the Sartorius Incucyte®), a high content imaging platform (e.g. Revvity Opera Phenix®, Molecular Devices ImageXpress®), organ-on-a-chip systems, or the various fluorescence microscopes, with or without live cell capabilities, depending on your application’s need for time-lapse or simple end-point measurements. Importantly, this means you can switch an assay between platforms according to your research’s changing needs with limited disturbance in the reagents used.

References:

Wlodkowic, D., .. & Darzynkiewicz, Z. (2013). Kinetic viability assays using DRAQ7 probe. Current protocols in cytometry, 65(1), 9-41.

Deo, P., .. & Lithgow, T. (2018). Outer membrane vesicles from Neisseria gonorrhoeae target PorB to mitochondria and induce apoptosis. PLoS pathogens, 14(3), e1006945.

Automated, Multi-Parameter, Kinetic Methods to Quantify Cell Death - Application Note. Brad Larson, Principal Scientist, BioTek Instruments, Inc., Winooski, VT USA. March, 2018

Jost, T., .. & Hecht, M. (2022). Influence of alectinib and crizotinib on ionizing radiation-in vitro analysis of ALK/ROS1-wildtype lung tissue cells. Neoplasia, 27, 100780.