Guava Technologies publishes a technical poster that describes results from a live cell multi-parameter fluorescence assay that may be used to measure mitochondrial membrane potential and apoptosis
Fluorescence-based assays designed to evaluate the functional status of mitochondria are emerging as useful tools to elucidate the role of mitochondrial activity in the apoptosis cascade, cell cycle and other cellular processes.
To undertake such measurements automated single-cell analysis is the technique of choice due to its sensitivity and high reproducibility.
In developing the MitoPotential Kit, Guava has produced an optimised assay format for 96-well plates that combines the sensitivity of a single-cell based method with the throughput and ease-of-use of a microplate reader.
The new technical poster describes how the Guava MitoPotential assay when used in conjunction with the Guava EasyCyte System, a blue laser 96-well microcapillary flow cytometry instrument, allows users to discern healthy, polarised cells from the apoptotic/depolarised and dead cells.
Loss of mitochondrial membrane activity is often observed to be associated with the early stages of apoptosis.
The Guava MitoPotential assay uses the fluorescent cationic dye, JC-1, and the dead cell dye, 7-AAD, for discerning apoptotic cells from dead cells.
In healthy, non-apoptotic cells, the JC-1 dye enters the mitochondrial matrix, accumulates as aggregates and stains the mitochondria bright red.
In apoptotic cells, the mitochondrial membrane potential collapses, and the JC-1 enters the cytoplasm as a monomeric form where it fluoresces green.
Results are automatically calculated and presented in a statistical format that is easy to interpret, with total cell counts and percentages for those cell populations that are either healthy, apoptotic, or dead.
In the poster the researchers demonstrate how they optimised the assay for JC-1 dye penetration and concentration and how the dye has different staining patterns, depending upon the cell line and apoptotic inducer used.
The poster also shows how apoptotic events were verified by comparing EC50 values from four well-known inducers of apoptosis with alternative Annexin V-FITC kit.
