Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.
Dysfunktionelle skeletmuskulaturen mitokondrier spille en rolle i ændret metabolisme observeret med aldring, fedme og type II diabetes. Mitokondriske respirometriske assays fra isolerede mitokondriske præparater gøre det muligt at vurdere mitokondriefunktionen, samt bestemmelse af mekanismen (r) for lægemidler og proteiner, som modulerer metabolisme. Aktuelle isoleringsprocedurer kræver ofte store mængder af væv til opnåelse af høje mitokondrier er nødvendige for respirometriske assays kvalitet. De metoder, der præsenteres heri beskriver, hvordan høj kvalitet oprenset mitokondrier (~ 450 ug) kan isoleres fra minimale mængder (~ 75-100 mg) af muse skeletmuskulatur til anvendelse i high throughput respiratoriske målinger. Vi konstateret, at vores isolation metode giver 92,5 ± 2,0% intakt mitokondrier ved måling citratsynthase aktivitet spektrofotometrisk. Desuden Western blot-analyse i isolerede mitokondrier resulterede i det svage ekspression af cytosoLIC protein, GAPDH og robust ekspression af mitokondrieprotein, COXIV. Fraværet af en fremtrædende GAPDH bånd i de isolerede mitokondrier er indikativ for lille forurening fra ikke-mitokondriske kilder i isolation procedure. Vigtigst, måling af O 2 forbrug sats med mikro-plade baseret teknologi og bestemme det respiratoriske kontrol ratio (RCR) for koblede respirometriske analyser viser stærkt koblede (RCR;> 6 for alle analyser) og funktionelle mitokondrier. Afslutningsvis, en markant reduktion motordrevet homogenisering hastigheden af en tidligere rapporteret fremgangsmåde tilsætning af et separat hakning trin og har givet isolationen af høj kvalitet og oprensede mitochondrier fra mindre mængder af muse skeletmuskulatur, der resulterer i stærkt koblede mitokondrier, der respirerer med høj funktion under mikrotiterplade-baseret respirometirc assays.
The primary function of mitochondria is to produce ATP from oxidative phosphorylation. However, mitochondria have many other important cellular functions including but not limited to: the production and detoxification of reactive oxygen species, the regulation of cytoplasmic and mitochondrial calcium, organelle trafficking, ionic homeostasis, and involvement in apoptosis1,2. Therefore, it is not surprising that dysfunctional mitochondria play a role in many disease pathologies, such as aging, neurodegenerative diseases, cardiovascular disease, cancer, obesity, and diabetes3,4. Importantly, skeletal muscle mitochondria specifically are involved in many of these pathologies3-5.
Mitochondrial respiration assays using isolated mitochondria allow for the assessment of electron transport chain and oxidative phosphorylation function, and the determination of mechanism(s) of action of drugs and proteins that modulate metabolism. Mitochondrial isolation procedures exist for multiple tissue and cell types for a variety of species6,7. However, these procedures often require large quantities of tissue/cells for a high quality mitochondria yield necessary for classic respirometric assays.
Microplate based respirometirc assays allow for high throughput measurements using minimal quantities of isolated mitochondria, often just several µg per well8. Therefore, we present a modification of previously published methods7 to allow for high quality mitochondria to be isolated from smaller quantities of mouse skeletal muscle for use in microplate based respirometirc assays. In addition, methods are provided to establish the quality of the mitochondrial isolation preparation and the integrity of the mitochondrial membranes. Given that skeletal muscle mitochondria are involved in many pathological conditions, the measurement of O2 consumption in mechanistically driven studies is becoming more prevalent in biomedical research9,10.
De metoder, der præsenteres heri give en detaljeret beskrivelse af et mitokondrie isolation proceduren fra minimale mængder (~ 75-100 mg) af mus skeletmuskulatur. Denne isolation procedure er i stand til at give høj funktion, ren mitochondrier (~ 450 mg) som det fremgår af O 2 forbrug priser, RCR værdier, maksimal citrat syntaseaktivitet og protein udtryk fra immunoblotting. Vigtigt er det, kan mitokondrierne isoleret fra denne fremgangsmåde bruges til flere respirometirc assays med mikrotiterplade-base…
The authors have nothing to disclose.
The Fralin Life Science Research Institute and The Metabolic Phenotyping Core at Virginia Tech supported this work.
Essentially Fatty | Sigma Aldrich | A6003 | N/A |
Acid Free- BSA | |||
Tris/HCl | Promega | H5123 | N/A |
KCL | Sigma Aldrich | P9541 | N/A |
Tris Base | Promega | H5135 | N/A |
EDTA | Sigma Aldrich | E6511 | N/A |
EGTA | Sigma Aldrich | E4378 | N/A |
Sucrose | Sigma Aldrich | S7903 | N/A |
D-Mannitol | Sigma Aldrich | 63559 | N/A |
Trypsin-EDTA (0.25%), phenol red | Thermo Scientific | 25200-056 | N/A |
Sodium Chloride White Crystals or Crystalline Powder ≥99.0 % |
Fisher Scientific | BP3581 | N/A |
Sodium dodecyl sulfate | Sigma Aldrich | L3771 | N/A |
Sodium deoxycholate | Sigma Aldrich | D6750 | N/A |
Polyoxyethylene (12) nonylphenyl ether, branched | Sigma Aldrich | 238651 | N/A |
Single Edge Razor Blades | Fisher Scientific | 12-640 | N/A |
Falcon- 100 uM Nylon Cell Strainers | Fisher Scientific | 352360 | N/A |
Halt Protease & Phosphatse Inhibitor Cocktail | Thermo Scientific | 1861284 | N/A |
1.5mL microcentrifuge tubes with screw cap | Thermo Scientific | 3474 | N/A |
Zirconium Oxide beads | Fisher Scientific | C9012112 | N/A |
GAPDH antibody (1D4) | Santa Cruz Biotechnology | sc-59540 | N/A |
Anti- COXIV antibody | Cell Signaling | 4844s | Any mitochondrial inner membrane protein will suffice |
Peroxidase conjugated affinipure Donkey, Anti Rabbit IgG (H+L) | Jackson ImmunoResearh | 711-035-152 | N/A |
Peroxidase conjugated affinipure Goat, Anti Mouse IgG (H+L) | Jackson ImmunoResearh | 115-001-003 | N/A |
Triton-X100 | Sigma Aldrich | X100 | N/A |
Pierce BCA Protein Assay Kit | Thermo Scientific | 23225 | N/A |
Pyruvic Acid, 98% | Sigma Aldrich | 107360 | Store at 4°C,pH to 7.4 with KOH prior to use in respirometric assay |
Succinic Acid | Sigma Aldrich | S9512 | Store at room temperature, pH to 7.4 with KOH prior to use in respirometric assay |
L(-) Malic Acid, BioXtra, ≥95% | Sigma Aldrich | M6413 | Store at room temperature, to 7.4 with KOH prior to use in respirometric assay |
L-Glutamic acid | Sigma Aldrich | G1251 | Store at room temperature, to 7.4 with KOH prior to use in respirometric assay, to 7.4 with KOH prior to use in respirometric assay |
Palmitoyl L-carnitine chloride | Sigma Aldrich | P1645 | Store at -20°C |
Oligomycin A, ≥ 95% (HPLC) | Sigma Aldrich | 75351 | Store at -20°C |
Carbonyl cyanide 4-(trifluoromethoxy) | Sigma Aldrich | C2920 | Store at 2-8°C |
phenylhydrazone | |||
≥98% (TLC), powder [FCCP] | |||
Antimycin A from streptomyces sp. | Sigma Aldrich | A8674 | Store at -20°C |
Adenosine 5′-diphosphate monopotassium salt dehydrate [ADP] | Sigma Aldrich | A5285 | Store at -20°C, to 7.4 with KOH prior to use in respirometric assay |
Rotenone | Sigma Aldrich | R8875 | Store at room temperature |