Isolation and Purification of Functional Platelet Mitochondria Using Discontinuous Percoll Gradient
Bibliographic record
Abstract
INTRODUCTION 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. 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. OBJECTIVES Evaluate the purity, integrity and yield of two different methods of isolation of mitochondria in human platelets. METHODS First, 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). RESULTS Data 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). CONCLUSIONS Results 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 Information Canadian Institutes of Health Research, New Brunswick Health Research Foundation, New Brunswick Innovation Foundation This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.002 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".