Isolation and Purification of Functional Platelet Mitochondria Using Discontinuous Percoll Gradient
Notice bibliographique
Résumé
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 .
Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.
Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,001 | 0,000 |
| Méta-épidémiologie (sens large) | 0,001 | 0,000 |
| Bibliométrie | 0,001 | 0,001 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,001 | 0,000 |
| Science ouverte | 0,001 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,002 | 0,002 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».