Isolering af mitokondrier fra minimale mængder af Mouse Skeletmuskel for High Throughput Microplate Respiratory Målinger

Summary

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.

Abstract

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.

Introduction

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 apoptosis^1,2. 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 diabetes^3,4. Importantly, skeletal muscle mitochondria specifically are involved in many of these pathologies^3-5.

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 species^6,7. 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 research^9,10.

Protocol

Dyreforsøg blev udført under en godkendt protokol af Institutional Animal Care og brug Udvalg på Virginia Polytechnic Institute og State University. 1. Opsætning (Tid: ~ 45 min) Optø frosne lagre af 0,25% trypsin, Isolation Buffer for mitokondrier (IBM) 1 og IBM2 i et 37 ° C vandbad. Skyl glasvarer og dissektionsinstrumenter i 70% ethanol efterfulgt af højvande renhed. Forbered 0,05% trypsin-opløsning fra 0,25% trypsin lager ved at fortynde 1 del trypsi…

Representative Results

Citrat syntaseaktivitet fungerer som et mål for membran integritet, da citrat syntase er beliggende i den indre mitokondrielle membran, og derfor ikke bør være til stede i suspensioner af mitokondrier med intakte membraner. Figur 1 repræsenterer citrat syntaseaktivitet i ikke-ultralydbehandlet mitokondrie prøver sammenlignet med lydbehandledes prøver fra samme isolation. Sonikering mitokondrierne resulterer i en statistisk signifikant stigning i citratsynthase aktivitet (p <0,01). Det er vigtig…

Discussion

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…

Materials

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