Rapid isolation and purification of functional platelet mitochondria using a discontinuous Percoll gradient

Abstract

The 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 but is also involved in many cellular processes. Of interest, mitochondria and mitochondrial components (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 mitochondrial isolation methods have been previously published, these techniques are aimed at yielding mitochondria from cells types other than platelets. In addition, little information is known on the number of platelet-derived microparticles that can contaminate the mitochondrial preparation or even the overall quality and integrity of the mitochondria. In this project, we provide an alternate purification method yielding mitochondria of high purity and integrity from human platelets. Using human platelets, flow cytometry and transmission electron microscopy experiments were performed to demonstrate that the Percoll gradient method yielded significantly purified mitochondria by removing platelet membrane debris. Mitochondrial respiration following the substrate-uncoupler-inhibitor-titration (SUIT) protocol was similar in both the purified and crude mitochondrial extraction methods. Finally, the cytochrome c effect and JC-1 staining did not exhibit a significant difference between the two methods, suggesting that the mitochondrial integrity was not affected. Our study suggests that the Percoll discontinuous gradient purifies viable platelet-derived mitochondria by removing platelet-derived debris, including microparticles, therefore confirming that this isolation method is ideal for studying the downstream effects of intact mitochondria in mitochondrial-related diseases.

Keywords:

• Mitochondria isolation
• mitochondria membrane integrity
• Percoll extraction method
• platelet-derived microparticles
• platelet-derived mitochondria

Author‘s contribution

Participated in research design: Jacob L. Léger, Nicolas Pichaud and Luc H. Boudreau.

Conducted experiments and performed data analysis: Jacob L. Léger, Jean-Luc Jougleux and Fanta Savadogo.

Wrote or contributed to the writing of the manuscript: Jacob L. Léger, Nicolas Pichaud, and Luc H. Boudreau.

Conflicts of interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.

Ethical approval

Blood was obtained from healthy consenting volunteers. This research project was approved by the Comité d‘éthique de la recherche avec les êtres humains at the Université de Moncton.

Additional information

Funding

J.L.L. is a recipient of a master‘s scholarship from the Canadian Institutes of Health Research (CIHR); J.L.J is a recipient of a New Brunswick Health Research Foundation (NBHRF) and Maritime SPOR Support Unit (MSSU) postdoctoral fellowship. L.H.B. would like to acknowledge the CIHR, NBHRF, the New Brunswick Innovation Foundation (NBIF) and the Université de Moncton. The work of N.P. is supported by grants from NSERC, NBIF and Université de Moncton.