पारगम्यता संक्रमण ताकना खोलने की पृथक माउस Mitochondria हार्ट में मल्टी पैरामीटर मापन
Summary
Mitochondrial पृथक माउस mitochondria दिल में पारगम्यता संक्रमण ताकना खोलने की माप के लिए एक spectrofluorometric प्रोटोकॉल को यहां प्रस्तुत किया है. परख mitochondria सीए के साथ माप शामिल<sup> 2 +</sup> हैंडलिंग, mitochondrial झिल्ली क्षमता और mitochondrial मात्रा. उच्च गुणवत्ता और कार्यात्मक दिल mitochondria प्राप्त करने के लिए प्रक्रिया भी वर्णित है.
Abstract
The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.
Protocol
Discussion
यहाँ प्रस्तुत प्रोटोकॉल आवश्यक प्रयोगात्मक पृथक mitochondria दिल (1 चित्रा और चित्रा 4) में पारगम्यता संक्रमण ताकना खोलने का आकलन चरणों का वर्णन: माउस दिल mitochondria के लिए अलग प्रक्रिया, श्वसन नियंत्रण …
Disclosures
The authors have nothing to disclose.
Acknowledgements
यह काम HL094536 (BJH) द्वारा समर्थित किया गया.
Materials
| Name of the reagent | Company | Catalogue number |
| Trypsin | Sigma-Aldrich | T3030 |
| Trypsin inhibitor (soybean) | Sigma-Aldrich | T9128 |
| Sodium hydrosulfite | Sigma-Aldrich | 71699 |
| Rotenone | Sigma-Aldrich | R8875 |
| Cytochrome c | Sigma-Aldrich | C7752 |
| Alamethicin | Sigma-Aldrich | A4665 |
| CCCP | Sigma-Aldrich | C2759 |
| Cyclosporin A | Calbiochem | 239835 |
| Fura FF | Invitrogen | F14180 |
| JC-1 | Invitrogen | T3168 |
| Tissue grinder Potter-Elvehjem with Teflon pestle 15 ml | Wheaton Industries | |
| Overhead stirrer | Wheaton Industries | 903475 |
| Oxytherm (temperature controlled oxygen electrode) | Hansatech Instruments | |
| QuantaMaster 80 dual emission spectrofluorometer | Photon Technology International, Inc. |
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