Isolation and Purification of Functional Platelet Mitochondria Using Discontinuous Percoll Gradient

Abstract

INTRODUCTIONThe isolation of mitochondria is gaining importance in experimental and clinical laboratory settings. The mitochondrion is known as the powerhouse of the cell as it produces the energy to power most cellular functions. However, mitochondria and its typical hallmarks (i.e. circular DNA, N‐formylated peptides, cardiolipin) have been involved in several human inflammatory pathologies, such as cancer, Alzheimer's disease, Parkinson's disease and rheumatoid arthritis. Therefore, stringent methods of isolation and purification of mitochondria are of the utmost importance in assessing mitochondrial‐related diseases. While several isolation kits are available commercially, they can be somewhat expensive and not suitable for some downstream applications. In this project, we provide an alternative purification method yielding mitochondria of high purity and integrity using human platelets.OBJECTIVESEvaluate the purity, integrity and yield of two different methods of isolation of mitochondria in human platelets.METHODSFirst, platelets were isolated from the blood of healthy donors. Then the brute fraction of platelet‐derived mitochondria was obtained using a potter homogenizer, followed by several differential centrifugation. To obtain the purified fraction, the mitochondrial extract was centrifuged on a discontinuous Percoll gradient (GE Healthcare). The purity of mitochondria was determined by flow cytometry (FC500, Beckman Coulter) using specific platelet marker anti‐CD41‐FITC (BioLegend), and by transmission electron microscopy (TEM). The respiratory capacity of mitochondria was measured by high‐resolution respirometry (Oroboros instruments). The total yield of mitochondria was determined by flow cytometry using MitoTracker Deep Red (Molecular Probes) and by the micro‐Smith method. Finally, the integrity of the mitochondrial membrane potential was assessed with JC‐1 staining (Molecular Probes).RESULTSData generated by flow cytometry shows that the Percoll gradient significantly purified mitochondria by removing 50% of platelet membrane debris (paired t‐test, p < 0.01). TEM analysis shows similar results. Mitochondrial respiration following the substrate uncouple inhibitor titration protocol is identical in purified and in brute mitochondria. Additionally, the cytochrome c effect is 5%, while JC‐1 staining shows no significant difference between methods suggesting integrity both in the inner and outer mitochondrial membrane. On the other hand, the mitochondrial protein yield was significantly decreased after purification (paired t‐test, p < 0.01).CONCLUSIONSResults of this study suggest that the Percoll discontinuous gradient purifies viable platelet‐derived mitochondria. Conversely, mitochondrial yield may be less important than obtained in other methods; however, it could be explained by the clustering of mitochondria containing less platelet debris. Relatively inexpensive, this method of purification is ideal for studying the downstream effects of intact mitochondria in mitochondrial‐related diseases.Support or Funding InformationCanadian Institutes of Health Research, New Brunswick Health Research Foundation, New Brunswick Innovation FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.