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. Author manuscript; available in PMC: 2021 Oct 21.
Published in final edited form as: Methods Cell Biol. 2019 Dec 10;155:3–31. doi: 10.1016/bs.mcb.2019.10.002

Isolation of mitochondria from cells and tissues

Pin-Chao Liao 1,*, Christian Bergamini 2,*, Romana Fato 2, Liza A Pon 1,^, Francesco Pallotti 3,^
PMCID: PMC8530414  NIHMSID: NIHMS1680511  PMID: 32183964

Abstract

Isolated mitochondria are useful to study fundamental processes including mitochondrial respiration, metabolic activity, protein import, membrane fusion, protein complex assembly, as well as interactions of mitochondria with the cytoskeleton, nuclear encoded mRNAs, and other organelles. In addition, studies of the mitochondrial proteome, phosphoproteome, and lipidome are dependent on preparation of highly purified mitochondria (Boldogh, Vojtov, Karmon, & Pon, 1998; Cui, Conte, Fox, Zara, & Winge, 2014; Marc et al., 2002; Meeusen, McCaffery, & Nunnari, 2004; Reinders et al., 2007; Schneiter et al., 1999; Stuart & Koehler, 2007). Most methods to isolate mitochondria rely on differential centrifugation, a two-step centrifugation carried out at low speed to remove intact cells, cell and tissue debris, and nuclei from whole cell extracts followed by high speed centrifugation to concentrate mitochondria and separate them from other organelles. However, methods to disrupt cells and tissue vary. Moreover, density gradient centrifugation or affinity purification of the organelle are used to further purify mitochondria or to separate different populations of the organelle. Here, we describe protocols to isolate mitochondria from different cells and tissues as well as approaches to assess the purity and integrity of isolated organelles.

Keywords: mitochondria, yeast, subcellular fractionation, affinity purification

Methods

Methods to isolate mitochondria from:

  1. Rat Brain by differential and density gradient centrifugation

  2. Rat Liver by differential centrifugation

  3. Beef Heart by differential centrifugation

  4. Skeletal muscle by differential centrifugation

  5. Cultured cells by differential centrifugation

  6. Budding yeast using magnetic beads

I. Isolation of Mitochondria from Rat Brain

Definition

Mitochondria from even for a single region of the brain are highly heterogeneous based on their morphological, histochemical, and enzymatic characteristics. Most methods are designed to isolate three distinct populations of mitochondria from rat brain: a) non-synaptic mitochondria, the so-called “free mitochondria” (FM); b) synaptosomal mitochondria (synaptic), which can be further subdivided in two fractions based on sedimentation properties, heavy (HM) and light (LM) (Reijnierse, Veldstra, & Van den Berg, 1975; Van den Berg, 1973). Synaptosomal mitochondria are involved in regulating neurotransmitter release and synaptic vesicle formation. In contrast, non-synaptic mitochondria derive from multiple cell types and from neuronal soma and are involved in microRNA regulation and energy production (Ly & Verstreken, 2006; Vos, Lauwers, & Verstreken, 2010; Wang, Sullivan, & Springer, 2017).

Rationale

The starting material for this preparation is usually dissected rat or mouse brain, sometimes pooled in 3 or 4 units from the same strain. Crude mitochondria are isolated from brain extracts by differential centrifugation. Different populations of mitochondria are prepared from crude mitochondria using discontinuous gradients (sucrose, Ficoll, or metrizamide).

Materials, equipment and reagents

Reagents

The pH of all solutions is checked daily.

  • Solution A: Sucrose 0.32 M, EDTA-K+ 1.0 mM, Tris-HCl 10 mM, pH 7.4

  • Solution B: Sucrose 0.32 M, EDTA-K+ 50 μM, Tris-HCl 10 mM, pH 7.4

  • Solution C: Tris-HCl 6 mM, pH 8.1

  • Solution D: Mannitol 0.24 M, Sucrose 60 mM, EDTA-K+ 50 μM, Tris-HCl 10 mM, pH 7.4

  • Solution E: Ficoll 3% (w/w), Mannitol 0.12 M, Sucrose 30 mM, EDTA-K+ 25 μM, Tris-HCl 5 mM, pH 7.4

  • Solution F: Mannitol 0.22M, Sucrose 0.07 M, Tris-HCl 50 mM, EDTA 1 mM, pH 7.2

Reagents for gradients
  • Gradient I:
    • Ficoll 7.5% (w/w) in solution B
    • Ficoll 12% (w/w) in solution B
  • Gradient II:
    • Ficoll 4.5% (w/w) in solution D
    • Ficoll 6% (w/w) in solution D
Equipment
  • For tissue homogenization
    • Potter-Elvehjem homogenizer consisting of a large clearance (0.1–0.15 mm, volume 10 mL) Teflon pestle driven into a glass vessel mechanically (Braun S homogenizer)
  • For differential centrifugation
    • Centrifuge: Beckman J2–21 or Sorvall RC-5B
    • Fixed Angle Rotor: Beckman JA-20 or Sorvall SS-34
    • Tubes: Polyallomer thick-wall, 10 mL
  • For gradients
    • Ultracentrifuge: Sorvall OTD65B or Beckman L5–50
    • Swinging bucket rotor: AH-650 or SW 50.1
    • Tubes: Polyallomer thin-wall, 5 mL

Protocols

Preparation of 2-step discontinuous gradients

Solutions for gradients are prepared just before the use and kept on ice. Gradients are prepared just before the use and kept in the refrigerator.

Gradient I:

  1. Place 1.9 mL of the 12% Ficoll solution in a 5 mL ultracentrifuge tube.

  2. Gently place 1.9 mL of the Ficoll 7.5% step on top of the 12% Ficoll step, using caution to avoid mixing of the steps.

  3. Store at 4°C.

Gradient II:

  1. Place 1.4 mL of the 6% Ficoll solution in a 5 mL ultracentrifuge tube.

  2. Gently place 2.6 mL of the 4.5% Ficoll solution on top of the 6% Ficoll step, using caution to avoid mixing of the steps.

  3. Store at 4°C.

Preparation of crude mitochondria from rat brain

All solutions used are at 0–4°C. Each step of homogenization is performed at 0–4°C.

  1. The procedure described below is based on preparation from 1 rat brain (ca. 120 mg). Rats are sacrificed using established guidelines of ethical procedures.

  2. Place rat brain in a prepared refrigerated box (0–4°C) within 15 sec of euthanization and separate hemicortexes from hippocampus and striatum.

  3. Rinse and place the hemicortexes (freed from blood and debris) in 2.5 mL of Solution A.

  4. Homogenize the tissue in a Teflon-glass homogenizer by 5 up-and-down passes of the pestle at 800 rpm for a maximum of 1 min.

  5. Transfer the homogenate to a pre-chilled centrifuge tube, resuspend residual cell extract in the homogenizer in 2.5 mL of solution A and transfer this material to the centrifuge tube.

  6. Centrifuge the homogenate by gradually increasing the centrifugation rate to 1,000 × g over a period of 4 min, with centrifugation at 1,000 × g for additional 11 sec. Transfer the supernatant to a fresh centrifuge tube and store on ice.

  7. Resuspend the pellet with 2 mL of Solution A and homogenize the resuspended pellet at 300 rpm for 60 sec and 3 passages of the pestle, and transfer the homogenate to a fresh centrifuge tube.

  8. Resuspend residual cell extract in the homogenizer in 2.0 mL of solution A and transfer this material to the centrifuge tube containing the homogenate from step 7.

  9. Centrifuge the homogenate as in steps 6–7 and pool the supernatant with the supernatant from the first round of homogenization.

  10. Carry out one more round of homogenization of the pellet as in steps 8–9.

  11. Centrifuge the 3 pooled supernatants at 15,000 × g for 20 min.

  12. The pellet obtained is crude mitochondria.

Preparation of synaptonemal LM and HM fractions and free mitochondria from crude mitochondria

  1. Discard the supernatant and resuspend the “crude” mitochondrial pellet from step 12 above (which contains non-synaptic and synaptic mitochondria) to a final volume of 0.7 mL in solution A and homogenize at 150 rpm and 3 passages of the pestle.

  2. Gently apply the suspension to gradient I using caution to avoid mixing the gradient steps.

  3. Centrifuge in a swinging bucket rotor at 73,000 × g for 24 min. Myelin and synaptosomal mitochondria are recovered in two bands. Free mitochondria (FM) are recovered in the pellet.

  4. To isolate synaptosomal mitochondria, discard the myelin band and recover the synaptosomal band at the interface of the 7.5–12% gradients with a Pasteur pipette and place in a centrifuge tube.

  5. Dilute the synaptonemal band with Solution A to a final volume of 5 mL and centrifuge at 15,000 × g for 20 min.

  6. To isolate free mitochondria (FM), resuspend the pellet obtained from gradient I in Solution F to a final volume of 0.6–0.8 mL. This is the final FM fraction.

  7. To isolate HM and LM fractions, resuspend the pellet obtained from step 5 in 5 mL of solution C and homogenize at 150 rpm for 2 min and 4 pestle strokes.

  8. Transfer the homogenate to a pre-chilled centrifuge tube and centrifuge the suspension at 14,000 × g for 30 min.

  9. Resuspend the pellet in 5 mL of solution C and centrifuge the suspension at 14,000 × g for 30 min.

  10. Resuspend the pellet in solution E to a final volume 0.6 mL.

  11. Gently apply the suspension to gradient II, using caution to avoid mixing.

  12. Centrifuge at 10,000 × g for 30 min. The HM fraction is in the pellet and the LM fraction is at the interface of the 4.5–6% steps.

  13. To obtain the LM fraction, discard the band at the interface of the 3–4.5% steps. Transfer the LM fraction in band at the 4.5–6% interface to a fresh centrifuge tube with a Pasteur pipette and dilute it in an equal volume of solution A.

  14. Centrifuge the suspension at 15,000 × g for 30 min.

  15. Discard the supernatant and resuspend the pellet in Solution F to a final volume of 0.6–0.8 mL. This is the final LM fraction.

  16. To obtain the HM fraction, resuspend the pellet obtained from gradient II in Solution F to a final volume of 0.6–0.8 mL. This is the final HM fraction.

  17. Store all fractions at −80°C.

Analysis and statistics

Protein content is determined by the Lowry method (Lowry, Rosebrough, Farr, & Randall, 1951). Expected yields of mitochondrial protein from different fractions are: ~5 mg/mL for FM; ~1.50 mg/mL for LM; ~1.60 mg/mL for HM, corresponding to 10.8% (FM), 2.0% (LM), and 2.4% (HM) of the homogenate protein. The method allows the preparation of highly purified mitochondrial fractions from cerebral cortex of rat brain suitable for respiratory chain enzymatic activities (Pallotti & Lenaz, 2007). To ensure NADH availability, mitochondrial fractions are usually pulse sonicated 5 times for 10 sec/min at 150W in an ice bath under nitrogen gas prior to measurement of enzymatic activity (Pallotti et al., 1998). Cytochrome content can be evaluated by using the method of Vanneste (Vanneste, 1966) and Nicholls (Nicholls, 1976). These mitochondrial preparations can be assayed for CoQ content by reversed phase HPLC analysis after extraction with methanol and light petroleum (Battino et al., 1995).

Related techniques

Some recent methods can be applied for the isolation of mitochondria from an entire hemisphere of neonatal mice (Wang et al., 2011). Recent methods for isolation of mitochondria from postmortem human brain for mtDNA analysis (Devall et al., 2015) and for isolation of mitochondria in CNS injury and neurodegeneration (Hubbard et al., 2019), using magnetic beads that are conjugated to antibodies against TOM22.

Pros and cons

Pros Cons
Highly purified mitochondria Relatively low yield
Conservation of biochemical and functional properties Time intensive
Allows for isolation of different populations of mitochondria, increasing selectivity and sensibility
High reproducibility
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Troubleshooting & Optimization

Problem Solution
Low yield Repeat homogenization of the low speed (1,000×g) pellet
Altered specific enzymatic activities (markers of subcellular fractions) Wash the “crude” mitochondrial pellet least 2–3 times to remove the microsomal contamination
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II. Isolation of Mitochondria from Rat Liver

Definition

Rat liver is a suitable source of mitochondria, as it is easily obtained and less difficult to homogenize compared to other tissues. Moreover, the yield of mitochondria is typically high because the hepatocytes account for 90% of the liver volume and are rich in mitochondria (about 1,000 per cell; 20% of the total cellular protein). Mitochondria isolated from rat liver (RLM) maintain a high degree of coupling for more than 3 hours after isolation and are normally used for biochemical studies, including polarographic measurements.

Rationale

To obtain the RLM, we followed the differential centrifugation method of Costantini et al. (Costantini, Petronilli, Colonna, & Bernardi, 1995) with some modifications. Rats are starved during the night before the experiment to lower endogenous fatty acid levels and the glycogen content of the liver. Animals are sacrificed and the liver is immediately transferred to ice-cold extraction buffer. The extraction buffer contains albumin to remove free fatty acids, which uncouple oxidative phosphorylation. The tissue is then minced with scissors and homogenized with a Teflon-glass Potter-Elvehjem homogenizer. After low speed centrifugation to remove nuclei and unbroken cells, the resulting supernatant is filtered with gauze and centrifuged twice at high speed. Finally, the pellets are resuspended in sucrose buffer. The protein content is determined by the biuret method (Gornall, Bardawill, & David, 1949).

Materials

  • Working solutions - all at 0–4°C

  • Extraction buffer: 0.25 M sucrose, 0.01 M Tris, 0.1 mM ethylene-bis (oxoethylenenitrilo) tetraacetic acid, EGTA; pH 7.4 with HCl; 0.4% bovine serum albumin (BSA)

  • Sucrose buffer: 0.25 M sucrose, 0.01 M Tris, adjusted to pH 7.4 with MOPS ((3-(N-morpholino)propanesulfonic acid

Equipment

  • Dissecting tools, scissors

  • Gauze

  • Potter-Elvehjem homogenizer (clearance 0.1–0.15 mm; 25-ml working volume)

  • High-speed refrigerated centrifuge with fixed-angle rotor (e.g. (RC5B Sorvall centrifuge, rotor SS34) and suitable polycarbonate tubes

Protocols

All materials should be pre-cooled and all steps should be performed at 0–4 °C.

  1. Starve a 150–175 gram male albino Wistar rat overnight and sacrifice by cervical dislocation. Remove the liver immediately and transfer into a beaker of ice-cold extraction medium. Wash briefly to remove any remaining traces of blood.

  2. Finely mince the liver using scissors.

  3. Wash the minced tissue in a beaker containing 50 ml ice-cold extraction buffer.

  4. Decant the medium and replace with 40 ml of fresh medium.

  5. Homogenize the tissue with a Potter-Elvehjem homogenizer; 8–10 passes with the homogenizer are required to ensure maximal cell disruption.

  6. Dilute the suspension to 60 ml of buffer for one liver and centrifuge at 650 × g for 10 min to remove nuclei and unbroken cells.

  7. Carefully filter the supernatant through 4 layers of gauze and centrifuge it at 17,000 × g for 10 min.

  8. Discard the supernatant and resuspend the mitochondrial pellet in extraction buffer without BSA, and centrifuge as above.

  9. Discard the supernatant and resuspend the pellet in sucrose buffer at a concentration ~50 mg/ml.

Safety considerations and standards

All protocols using live animals must conform to governmental regulations regarding the care and use of laboratory animals.

Analysis and statistics

Typical yields for this procedure are 10 grams wet weight of mitochondria per rat liver. When energized with glutamate/malate, the mitochondria exhibit a 6–10 fold increase in oxygen consumption after the addition of ADP, which indicates that the organelles are largely intact. The respiratory activity assessed by the P:O ratios (about 3 for glutamate and 2 for succinate) approach theoretical values.

Related techniques

RLMs can be the starting material to obtain sub-mitochondrial particles (SMPs) through sonication. SMPs do not exhibit oxidative phosphorylation activity. However, since intact mitochondria are not permeable to NADH, SMPs are a useful model to study NADH-dependent activities. The method used in our experiments is essentially the one described by Gregg (Gregg, 1967). Mitochondria are prepared from 20 to 30 g of rat liver and the buffer used is the same as that used for the preparation of broken SMP particles from BHM (Lee & Ernster, 1967), with the addition of 0.4% BSA from the beginning of the isolation procedure.

Pros and cons

Pros Cons
Ready availability of rat liver to most laboratories Low purity
Mild homogenization conditions compared to other tissues
Rapid preparation
High yield
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Alternative methods/procedures

RLM can be further purified using density gradients. The method described by Reinhart et al. (Reinhart, Taylor, & Bygrave, 1982) allows one to obtain functionally intact and relatively uncontaminated mitochondria using a discontinuous Percoll gradient. An alternative method for obtaining highly purified mitochondria uses semi-automated cell rupture, termed pump-controlled cell rupture system (PCC). Upon subsequent purification steps, the mitochondrial fraction meets the quality and purity required for molecular analyses, e.g. proteomic comparisons or determination of diverse enzymatic activities (Schmitt, Eberhagen, Weber, Aichler, & Zischka, 2015).

Troubleshooting & Optimization

Problem Solution
Low quality of mitochondria The animals are normally deprived of food overnight to reduce the glycogen content of the liver; this facilitates the separation process.
Low respiratory control The clearance between the glass wall and the side of a pestle must be 0.3 – 0.5 mm. Use of a pestle that is too tight, or excessive homogenization, will damage the mitochondria.
High yields and quality also depend on the gentle and rapid handling of the tissue. For a whole liver, divide the starting material into 2–3 portions and homogenize them separately.
Removal of this lipid is essential, as free fatty acids are potent uncouplers.
Low purity of isolated mitochondria Contamination with other organelles can be reduced by washing the mitochondrial pellet gently.
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III. Isolation of Mitochondria from Beef Heart

Definition

The bovine heart is useful for the isolation of mitochondria for two reasons. First, heart muscle tissue can be obtained in large amounts, which can yield several grams of mitochondrial proteins from a single isolation procedure. Second, heart muscle mitochondria do not exhibit the age-associated declines in function observed in rat liver mitochondria (Azzone, Colonna, & Ziche, 1979). Here, we described a method, modified from the original paper by Smith (Smith, 1967), to isolate bovine heart mitochondria (BHM) that have good enzymatic activity and are stable up to one year when stored at −20°C.

Rationale

Differential centrifugation is the most widely used method for obtaining large amounts of mitochondria for respiratory studies in a relatively short time. The method is often preferred to gradient centrifugation because it avoids the use of long density gradients, which can expose the mitochondria to potentially damaging g forces and unsuitable buffers (Graham, 2001). To isolate BHM, hearts must be removed from animals immediately after slaughter and kept on ice during transport to the laboratory. The hearts are then cut roughly and ground with a commercial meat grinder. After this phase, the ground tissue is washed and homogenized with a blender. Once homogenized, the suspension is centrifuged at low speed to remove intact cells and nuclei, and then centrifuged again at high speed to obtain a mitochondrial pellet. All operations must be carried out in a cold room at 4°C and the pH constantly adjusted to 7.8 with Tris base solution.

Materials

One or two beef hearts (approx. 2–2.5 Kg) freshly isolated and stored in ice.

Working solutions

  • Washing solution: 0.15M KCl

  • Sucrose buffer: 0.25M sucrose, 10mM Tris, pH 7.6 with HCl

  • 1M Tris base

Equipment

  • Commercial meat grinder

  • Waring blender

  • Cheesecloth

  • Teflon/glass Potter-Elvejhem homogenizer (clearance: 0.1–0.15 mm; volume 30 mL)

  • Low-speed centrifuge with fixed angle rotor or swinging bucket rotor and 250- to 750-ml bottles

  • High-speed centrifuge with fixed-angle rotor and 50 mL polycarbonate tubes

  • Cold room

Protocol

All subsequent procedures are carried out at 4°C.

  1. Freshly isolated bovine hearts are cleaned of connective and fat tissues and cut into small cubes.

  2. Tissue is washed with washing solution (approx. 2.5 litres), passed through a commercial meat grinder, and resuspended in sucrose buffer (approx. 200 ml of sucrose buffer for 100 g of tissue)

  3. The suspension is homogenized for 60 s at high speed in a Waring blender (two 30-s pulses with a 10-s pause between pulses). The pH of the suspension must be adjusted to 7.5 with 1M Tris.

  4. The homogenate is centrifuged for 10 min at 1,000 × g to remove unruptured muscle tissue and nuclei.

  5. The supernatant is filtered through two layers of cheesecloth to remove lipid granules and then centrifuged for 30 min at 26,000 × g.

  6. The mitochondrial pellet obtained is resuspended in 4 volumes of sucrose buffer, homogenized with a Teflon/glass Potter-Elvehjem homogenizer (10 strokes using large clearance pestle) and then centrifuged at 26,000 × g for 30 min.

  7. The pellet is resuspended in the sucrose buffer (approx. 40–50 mL) and stored at −80°C, at a protein concentration of 40 mg/ml.

Analysis and statistics

The average yield of intact BHM is approximately 1 mg protein per gram of starting mince. The protein content is determined by the biuret method (Gornall et al., 1949). The purity of the preparation is assessed spectrophotometrically by cytochrome content assay (Azzone et al., 1979). Intact beef heart mitochondria should possess a cytochrome aa3 content of 0.5 μmol/g protein and a cytochrome ratio c1/c/aa3 of 0.5:1:1. BHM have a P:O ratio in the presence of 300 μM of ADP (state 3 of respiration) of about 3 using pyruvate/malate as a substrate and about 2 in the presence of succinate, with a rate of substrate oxidation of 0.15–0.30 nAtoms of oxygen/min/mg protein and 0.05–0.09 nAtoms of oxygen/mg protein, respectively (Azzone et al., 1979; Smith, 1967). The respiratory control, expressed as the ratio between state 3 of respiration and state 4 respiration (without ADP), ranges between 3 and 6 with pyruvate-malate and between 2 and 3 in the presence of succinate.

Related techniques

BHM preparation obtained with the method described above can be used for the preparation of submitochondrial particles (SMP) (Lee & Ernster, 1967) and coupled submitochondrial particles (ETPH) (Beyer, 1967a). SMP are broken membrane fragments with good activity of enzymes such as NADH-coenzyme Q (CoQ) oxidoreductase and ubiquinol-cytochrome c oxidoreductase that are used for the kinetic characterization of Complexes I and III, respectively (Estornell, Fato, Pallotti, & Lenaz, 1993; Fato et al., 1993). ETPH are fully capable of undergoing coupled oxidative phosphorylation (Beyer, 1967a; Hansen & Smith, 1964). Moreover, BHM are a starting material for the preparation of R4B, a crude mitochondrial fraction enriched in Complex I and Complex III (Rieske, 1967). The R4B fraction can be further purified to obtain isolated Complex I, Complex III, Complex IV, and ATPase (Beyer, 1967b; Hatefi, 1978).

Pros and cons

Pros Cons
High yield
Inexpensive procedure
High stability of the mitochondria
Does not require ultracentrifugation		 Low purity
Need for suitable equipment for large volumes of buffers and a cold room
Time consuming procedure
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Troubleshooting and optimization

Problem Solution
Low P:O ratio Distilled water used for the isolation media and for the assay reagents must be of the highest quality
Resuspend the pellets more gently
The temperature must be maintained at 4°C during the entire isolation procedure.
Low purity The mitochondrial pellet has 2 layers, an upper part (light brown mitochondria) and a lower part (dark brown mitochondria). Light brown mitochondria are usually less pure, with lower P:O ratio and with less cytochromes aa3, and can be discarded.
Low yield Homogenization is not sufficient. Use more buffer during the homogenization and increase the time.
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IV. Isolation of Mitochondria from Skeletal Muscle

Definition

Biochemical analysis of mitochondria isolated from muscle tissue gives valuable information on pathological and physiological characteristics of human diseases related to impaired mitochondrial metabolism. Mitochondrial bioenergetics can be carried both on permeabilized muscle cells or fibers and on isolated mitochondria. Skeletal muscle mitochondria consist of two separate subpopulations, subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria, with different biochemical properties (Cogswell, Stevens, & Hood, 1993). The optimal isolation buffer for this preparation, isotonic nonionic solutions containing sucrose vs. nearly-isotonic ionic KCl-containing medium, has been a matter of debate. Overall, the latter isolation buffer is considered more suitable for isolation of skeletal muscle mitochondria because skeletal muscle tissue homogenized in isotonic sucrose has a gelatinous consistency (Estabrook, 1967; Pallotti & Lenaz, 2007). Tissue is limited (< 1 g whole tissue) for preparation of mitochondria from human or mouse model skeletal muscle, which can lead to low recovery of mitochondria (20–30%, compared to up to 40–50%, as reported only by Rasmussen et al. (Rasmussen, Andersen, & Rasmussen, 1997)).

Rationale

The majority of methods utilized for the isolation of the entire mitochondrial population from skeletal muscle are based on the differential centrifugation technique. Several basic methods are available, most of which were reviewed in the previous edition of this chapter (Pallotti & Lenaz, 2007). Other methods published recently use different centrifugation methods (Bharadwaj et al., 2015; Djafarzadeh & Jakob, 2017; Garcia-Cazarin, Snider, & Andrade, 2011; Lai et al., 2019). The protocol here described is suitable for standard biochemical and functional assays. It is optimized for low amounts of starting material (< 1 g muscle) and uses isotonic sucrose under conditions where the homogenate does not have a gelatinous consistency.

Materials, equipment and reagents

  • Isolation Buffer: Mannitol 0.22M, Sucrose 0.007M, Tris 2mM, 1 mM EDTA, 20 mM HEPES, pH7.2

  • 0.4% albumin

  • Trypsin (or Nagarse) 0.3 mg/mL

Equipment

  • Potter-Elvehjem homogenizer consisting of a large clearance (0.1–0.15 mm, volume 10 mL) Teflon pestle driven into a glass vessel mechanically (Braun S homogenizer)

  • High speed centrifuge: Beckman J2–21 or Sorvall RC-5B

  • Fixed angle rotor: Beckman JA-20 or Sorvall SS-34

  • Centrifuge Tubes: Polycarbonate, 10 mL

Protocols

All procedures should be performed on ice; all consumables and tubes should be pre-chilled in ice; centrifuge should be set at 4°C.

  1. 100 mg of muscle tissue (freed form collagen and nerves).

  2. Place the tissue in Isolation buffer (approx. 1 mL) containing 0.4% albumin and mince in small pieces.

  3. Incubate the minced tissue in Isolation buffer (+ albumin 0.4%) with trypsin 0.3 mg/mL (100 mg tissue in 0.5 mL) for 5 min at RT, then place immediately on ice.

  4. Dilute in 10 volumes of Isolation buffer (+ albumin 0.4%) without trypsin.

  5. Homogenize the tissue (10 strokes using large clearance pestle).

  6. Centrifuge at 800 × g for 10 min.

  7. Collect the supernatant.

  8. Resuspend the crude nuclear fraction in the pellet in 2.5 mL of Isolation buffer with albumin and homogenize the solution (10 strokes using large clearance pestle).

  9. Centrifuge at 800 × g for 10 min.

  10. Collect the supernatant.

  11. Combine the supernatants obtained from steps 7 and 10.

  12. Centrifuge at 17,000 × g for 10 min.

  13. Wash the mitochondrial pellet twice in 2.5 mL of Isolation buffer and centrifuge at 17,000 × g for 10 min.

  14. Resuspend the pellet in 50–100 μL of the same buffer.

Expected yield: approx. 0.5 mg of mitochondrial protein from 100 mg of muscle tissue.

Related techniques

Alternative techniques are optimized for different amounts of starting material or applications. The method described by Djafarzadeh and Jakob is designed for the isolation of mitochondria from 5–10 g of skeletal muscle (Djafarzadeh & Jakob, 2017). Lai and colleagues published a method for isolation of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria (Lai et al., 2019).

Pros and cons

Pros Cons
Fast method Respiratory experiment should be performed within 1 hr of isolation
Inexpensive and very efficient procedure Force and speed of the pestle in the homogenization step can vary between operators
Mitochondria can be used for respirometry assays
Minimal quantities of skeletal muscle required
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Alternative methods/procedures

Mitochondrial respiration in skeletal muscle can be measured in permeabilized muscle fibers (Gnaiger, 2009) by using Oxygraphy for High-Resolution Respirometry (HRR) or directly on muscle homogenates (Pecinova, Drahota, Nuskova, Pecina, & Houstek, 2011).

Troubleshooting & Optimization

Problem Solution
Low yield Modify the ratio tissue:isolation medium (the ideal ratio is 1:5 to 1:10); make sure to perform all the isolation procedures in ice
Low quality of isolated mitochondria Remove the “fluffy” layer (which contains broken mitochondria and fragmented mitochondrial membranes) of the mitochondrial pellet. Further purify the dark brown pellet containing intact mitochondria by using discontinuous gradient (bearing in mind that some material can be lost by using this step).
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V. Isolation of Mitochondria from Cultured Cells

Definition

Human cultured cells represent a valid experimental model for investigating mitochondria function, both in physiological and pathological states. In particular, the transformed cells are suitable for the isolation of mitochondria, as they are easily cultured and can be obtained in large amounts. Mitochondria can also be isolated from primary cell cultures (e.g. fibroblasts).

Rationale

The method described here is a modification of Yang et al. (Yang et al., 1997). The yield varies with the type of cell line used. For examples, yield is typically high from HeLa cells, which are rich in mitochondria.

Two days before preparation, seed cells in tissue culture dishes. Cultured cells are detached from dishes by trypsinization and concentrated by centrifugation. The cell pellet is washed and cells are homogenized using a Potter-Elvehjem homogenizer. The mitochondria are isolated by differential centrifugation and the protein content determined by the biuret method (Gornall et al., 1949).

Materials

  • Cell pellet derived from tissue culture cells (approx. 5 × 106 ‒ 1 × 107 cells)

  • Extraction buffer: 0.25 M sucrose, 20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM PMSF

  • Phase contrast microscope

  • Refrigerated bench top centrifuge

  • Teflon/glass Potter-Elvehjem homogenizer (clearance 0.1–0.15 mm; volume 5 mL)

Protocol

Glassware and buffer must be precooled in an ice-bath. Steps 3–9 must be performed at 0–4°C to minimize the activation of damaging phospholipases and proteases.

  1. Remove the medium from the cells and wash the cells once with PBS.

  2. Remove PBS and detach the cells by suitable trypsinization condition

  3. Collect the cells by centrifugation (300 × g for 3 min).

  4. Wash the cells once with PBS and centrifuge, as above.

  5. Discard the supernatant and resuspend with 5 volumes of extraction buffer.

  6. The cellular suspension is homogenized with a Teflon-glass homogenizer with 10–20 up-and-down passes of the pestle.

  7. The homogenate is then centrifuged at 750 × g for 10 min.

  8. The resulting supernatant is transferred to a pre-chilled centrifuge tube and stored on ice, and the pellet is resuspended in extraction buffer, homogenized and centrifuged as for steps 5–7.

  9. The supernatants obtained from the two low-speed spins are pooled and then centrifuged at 10,000× g for 15 min. Crude mitochondria, which are recovered in the pellet, are resuspended in extraction buffer (approx. 20–25 μL).

Analysis and statistics

The crude mitochondria isolated using this method can be used for polarographic determinations, spectrophotometric determinations, and protein analysis, such as Western blots and ELISA. The yield depends on the type of cells and can vary from 7 to 50 μg of mitochondrial protein per 106 cells. For experiments requiring a high degree of purity (e.g. in vitro protein synthesis and proteomic studies), separation methods such as gradient centrifugation must be used (see related techniques section). The purity and the enrichment of mitochondria from whole cells can be assessed by Western blot analyses, measuring cytosolic (e.g. actin), nuclear (e.g. lamine A/C), and mitochondrial (e.g. OXPHOS subunits and porin) marker proteins. The integrity of the mitochondrial fraction can be tested by quantitation of an intermembrane space protein (e.g. cytochrome c) or a matrix proteins (e.g. cyclophilin D) by immunoblot or by measuring ADP stimulation by polarography. Alternatively, measuring mitochondrial Ca2+ buffering capacity is a fast and efficient method to assess the integrity of isolated mitochondria (Wettmarshausen & Perocchi, 2017).

Related techniques

Mitochondria of higher purity can be prepared using a sucrose gradient. A valid method, which is a modification of the “two-step” procedure described by Tapper et al. (Tapper, Van Etten, & Clayton, 1983), has been described by Magalhaes et al. (Magalhaes, Andreu, & Schon, 1998). An alternative method to mechanical homogenization of cells is nitrogen cavitation (Gottlieb & Adachi, 2000; Simpson, 2010). Mitochondria prepared by this method possess an intact outer membrane, high respiratory control ratios, and retain intermembrane space components, including cytochrome c and adenylate kinase.

Pros and cons

Pros Cons
Rapid method: the entire protocol takes about 90 min. The integrity of mitochondria must be checked carefully after isolation
Cultured cells are standardized models and easily obtained All the experiments that require intact mitochondria must be performed within 3 hrs after isolation
The protocol for mitochondria isolation must be adapted depending on cell type.
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Alternative methods/procedures

Mitochondria can be isolated from mammalian cells or tissues through the use of magnetic beads that are covalently coupled to antibodies that recognize mitochondrial outer membrane proteins (e.g. Tom20) (Hornig-Do et al., 2009). However, this method may alter mitochondrial ultrastructure or cover epitopes in the outer membrane (see section VI in this chapter).

Troubleshooting & Optimization

Problem Solution
Low yield Homogenization has a large impact on yield of the mitochondrial preparation.
We recommend use of a teflon-glass homogenizer. Homogenization using glass-glass homogenizer can lead to increased yield but can also damage mitochondria.
Visualization of cell disruption using a microscope can be used to optimize homogenization conditions for different cell lines. The homogenization step should lyse about 80% of the cells.
Fibroblast cell membranes are difficult to break open. To promote homogenization, freeze-thaw cells once harvested.
Low quality Homogenization and all the subsequent steps of the protocol must be performed at 4°C to minimize the activation of phospholipases and proteases.
Excessive homogenization can cause damage to the mitochondrial membrane and trigger release of mitochondrial components.
Avoid diluting the mitochondria with buffer. Mitochondria retain their function for a longer period, probably as a result of less exposure to oxygen, when stored in a concentrated form (30–50 mg/mL).
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VI. Affinity purification of mitochondria from yeast using magnetic beads

Definition

Crude mitochondria isolated from budding yeast by differential centrifugation contain other membrane or organelle contaminants (Daum, Böhni, & Schatz, 1982). While crude mitochondria can be purified further by density gradient ultracentrifugation (Boldogh & Pon, 2007; Glick & Pon, 1995), these methods require large amounts of starting material and access to an ultracentrifuge. Here, we describe a method to isolate highly purified mitochondria from yeast using magnetic bead affinity purification (Liao, Boldogh, Siegmund, Freyberg, & Pon, 2018). This method includes the removal of magnetic beads from mitochondria, therefore minimizing their interference in subsequent structural or functional studies.

Rationale

We tagged the mitochondrial outer membrane protein Tom70 at its chromosomal locus with 6xHis. Yeast expressing Tom70–6xHis are grown to mid-log phase in liquid medium containing a non-fermentable carbon source (lactate), which promotes mitochondrial biogenesis. Cells expressing Tom70–6xHis are then treated with zymolyase, which catalyzes breakdown of the cell wall, and the resulting spheroplasts are disrupted by Dounce homogenization. The cell lysates are subjected to low speed centrifugation to sediment intact cells, nuclei, and cellular debris, and the resulting supernatant - the mitochondria-enriched fraction - is incubated with Ni-NTA magnetic beads, Mitochondria that are bound to the beads are separated under magnetic fields, washed, and released from magnetic beads with imidazole treatment (Fig. 1A).

Figure 1. Isolation of mitochondria using Ni-NTA magnetic beads.

Figure 1.

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(A) Scheme of mitochondrial isolation using magnetic beads. Detailed steps are described in the main text. (B) Marker proteins for mitochondria (Tom70, Porin, cytochrome b2 [Cyb2], α-ketoglutarate dehydrogenase [Kgd1]), cytosol (hexokinase [Hxk]), ER (Sec61), and vacuole (Nyv1) are probed using Western blot analysis. Total protein load was assessed using TCE. (C) Crude or bead-purified mitochondria are treated with (+) or without (−) 100 μg/ml proteinase K (PK) for 30 min at 4°C. Mitochondrial outer membrane proteins (Tom70 and Porin) and an intermembrane space protein (Cyb2) are probed using Western blot analysis.

Materials, equipment and reagents

Strains

  • PLY31: MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 TOM70–6xHis-KanMX6

Materials

  • Zymolyase 20T (Seikagaku Corporation, Tokyo, Japan)

  • HisPur™ Ni-NTA Magnetic Beads (88831, Thermo Scientific, Grand Island, NY)

Stock solutions

  • 1 M Tris–SO4, pH 9.4. Autoclave.

  • 1 M DTT. Dissolve in water. Store at −20 °C.

  • 1 M MgCl2. Autoclave.

  • 2.4 M Sorbitol. Autoclave.

  • 200 mM Phenylmethylsulfonylfluoride (PMSF) (Roche, Indianapolis, IN). Dissolve in 100% ethanol. Store at −20 °C.

  • 1 M HEPES–KOH, pH 7.4. Adjust pH with KOH. Autoclave.

  • 1 M KPi, pH 7.4. (19.8 ml 1M KH2PO4 and 80.2 ml 1M K2HPO4) Autoclave.

  • Protease Inhibitor (PI) cocktails (all reagents are from Sigma, St. Louis, MO).

  • PI-1: 1000x stock dissolved in H20 (Sigma catalog numbers in parentheses): 0.5 mg/ml Pepstatin A in dimethyl sulfoxide (DMSO) (P4265), 0.5 mg/ml Chymostatin in DMSO (C7268), 0.5 mg/ml Antipain (A6191), 0.5 mg/ml Leupeptin (L2884), 0.5 mg/ml Aprotinin (A1153)

  • PI-2: 1000x stock dissolved in 100% ethanol: 10 mM Benzamidine–HCl (B6506), 1 mg/ml 1,10-Phenanthroline (P9375)

  • Both cocktails are divided into 0.5 ml aliquots and stored at −20 °C.

Working solutions

  • Lactate medium: 3 g/L Yeast extract (Difco), 0.5 g/L Glucose, 0.5 g/L CaCl2 2H2O, 0.5 g/L NaCl, 0.6 g/L MgCl2 6H2O, 1 g/L KH2PO4, 1 g/L NH4Cl, 22 ml/L of 90% Lactic acid, 7.5 g/L NaOH, adjust pH to 5.5 with NaOH pellets.

  • Tris–DTT buffer: 0.1M Tris–SO4, pH 9.4, 10 mM DTT

  • SP buffer: 1.2 M Sorbitol, 20 mM KPi, pH 7.4

  • SEH buffer (Add PI-1, PI-2, and PMSF immediately before use): 0.6 M Sorbitol, 20 mM HEPES–KOH, pH 7.4, 2 mM MgCl2, 1x PI-1, 1x PI-2, 1 mM PMSF

Equipment

  • Sorvall RC5C Refrigerated Centrifuge with Sorvall GS-3 rotor and Sorvall SS34 rotor

  • New Brunswick Innova 4330 Refrigerated Incubator Shaker

  • 40-ml glass/glass Dounce homogenizer (Wheaton Science Products, Millville, NJ)

  • 6-Tube Magnetic Separation Rack (New England Biolabs, Ipswich, MA)

  • Multipurpose tube rotator (Fischer Scientific, Hampton, NH)

Protocols

  1. Prepare precultures by inoculating a colony of yeast expressing 6xHis-tagged Tom70 into 5 ml of lactate medium in a 50-ml Falcon tube and incubate at 30°C with aeration (200 rpm in a New Brunswick shaking incubator) overnight. For 1.5-L growths prepare 3 precultures.

  2. Inoculate 1.5 L lactate medium in a 4.5-L shake flask with the 15 ml preculture. Grow yeast cells to mid-log phase at 30°C overnight with aeration, as above.

  3. Concentrate cells by centrifugation at 1500 × g for 5 min at 4°C (3,500 rpm in a Sorvall GS-3 rotor).

  4. Discard the supernatant and wash cells by resuspending the cell pellet with 200 ml water and concentrating them by another round of centrifugation at 1,500 × g for 5 min at 4°C. Remove the supernatant and weight the “wet” cell pellet. Typically, we obtain about 4 g wet cells in from a 1.5L culture.

  5. Resuspend the cell pellet in 20 ml of Tris–DTT buffer in a 125-ml Erlenmeyer flask, and incubate for 15 min at 30°C with shaking (200 rpm in a New Brunswick shaking incubator). Transfer the suspension to Sorvall SS34 rotor tubes, and centrifuge at 1,500 × g for 5 min at 4°C.

  6. Resuspend the cell pellet in 20 ml of SP buffer, and centrifuge at 1,500 × g for 5 min at 4°C. To convert yeasts to spheroplasts (cells without cell walls), resuspend cells in 20 ml of SP buffer containing Zymolyase 20T (7.5 mg/g yeast wet) and incubate in a 125-ml Erlenmeyer flask at 30°C for 40 min with shaking (200 rpm in a New Brunswick shaking incubator).

  7. Transfer the spheroplast suspension to a SS35 Sorvall tube and centrifuge at 4,500 × g (6,000 rpm in a Sorvall SS34 rotor) for 5 min at 4°C. Resuspend the pellet in 20 ml of ice-cold SEH buffer. Keep everything on ice in the following steps.

  8. Centrifuge the mixture at 4,500 × g for 5 min at 4°C. Resuspend the pellet of spheroplasts in 20 ml of ice-cold SEH buffer, and transfer to a prechilled 40-ml glass/glass Dounce homogenizer. Using the tight-fitting pestle, homogenize the spheroplasts with 15 forceful strokes.

  9. Centrifuge the homogenate at 1,500 × g for 5 min at 4°C. Collect the supernatant and centrifuge at 12,000 × g for 10 min at 4°C. Resuspend the pellets in 4 ml ice-cold SEH buffer. We refer to this fraction as the “mitochondria-enriched fraction.”

  10. Prepare HisPur™ Ni-NTA Magnetic Beads, as follows. Transfer 100 μl beads to a 1.5-ml microcentrifuge tube (100 μl beads/1 ml mitochondria-enriched fraction). Place the tube in a Magnetic Separation Rack for 30 sec to concentrate beads at the lateral surface of the tube adjacent to the magnet. Remove the residual liquid using a micropipette. Remove the tube from the separation rack, and wash beads with 500 μl SEH buffer 2 times.

  11. Incubate 1 ml of mitochondria-enriched fraction with washed beads for 10 to 60 min at 4°C with rotation (low speed) in a multipurpose tube rotator. Place the mixture in the separation rack for 1 min to separate the magnetic bead-bound mitochondria from other organelles or debris. Wash the bead-bound mitochondria 3 times with 15 mM imidazole in ice-cold SEH buffer.

  12. To elute mitochondria, incubate magnetic bead-bound mitochondria with 50 μl of 500 mM imidazole in ice-cold SEH buffer for 5 min with rotation. Place the tube in the magnetic field to trap the beads. Transfer the released mitochondria to a microcentrifuge tube and centrifuge at 12,000 × g for 5 min at 4°C. Remove the supernatant and resuspend the mitochondrial pellet in ice cold SEH buffer, as needed.

Analysis and statistics

Examination of mitochondrial purity

We typically obtain about 100 μg of mitochondria from 1 g of wet yeast after incubation of the mitochondria-enriched fraction with the magnetic beads for 60 min. The yield obtained after 10-min incubation is reduced by 25% compared to that obtained after a 60-min incubation.

To compare the purity of mitochondria preparations using magnetic beads and differential centrifugation, we perform Western blot analysis using antibodies against marker proteins of the mitochondrial outer membrane (Tom70 and porin), intermembrane space (cytochrome b2, Cyb2), matrix (α-ketoglutarate dehydrogenase, Kgd1), ER (Sec61), cytosol (hexokinase), and vacuole (the vSNARE Nyv1). Mitochondrial proteins, Tom70, porin, Cyb2, and Kgd1, are expected to enrich in crude and magnetic bead-purified mitochondria. In addition, cytosolic, ER, and vacuole contamination in bead-purified mitochondria are expected to be lower compared to that observed in crude mitochondria (Fig. 1B). These indicate that mitochondria isolated using the magnetic bead method are purer compared to crude mitochondria prepared by differential centrifugation.

Characterization of mitochondrial integrity

To characterize the integrity of mitochondria, we treat them with proteinase K. If mitochondria are intact, outer membrane proteins (Tom70) are expected to be protease-sensitive, and the intermembrane space protein (cytochrome b2) is expected to be protease-protected. Porin, an outer membrane protein that is protease-resistant is used as a protein loading control. (Fig. 1C).

Related techniques

Other magnetic bead-based isolation methods are included as follows. Magnetic beads coated with antibodies have been used to isolate mitochondria in mammalian systems from cells (Hornig-Do et al., 2009), mouse tissues (Franko et al., 2013), and human cortex (Khattar et al., 2016). Lipophilic and delocalized triphenyl phosphonium (TPP) cation-bound magnetic beads, which recognize mitochondria by mitochondrial membrane potential (Δψ), have also been applied to isolate mitochondria (Banik, Askins, & Dhar, 2016; Banik & Dhar, 2017). However, mitochondria isolated using these methods cannot be released from magnetic beads, which may affect mitochondrial ultrastructure and/or cover epitopes on the mitochondrial outer membrane.

Pros and cons

Pros Cons
Generates highly pure mitochondria Not suited for large amounts of mitochondrial preparation
Less time-intensive compared to other methods for preparation of highly purified mitochondria
Mitochondria are released from magnetic beads
Does not require large amount of starting materials
Does not require ultracentrifugation
More cost-effective than commercially-available magnetic bead purification kits
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Troubleshooting & Optimization

Problem Solution
Low yield Use lactate medium. Other yeast growth medium may lower the yield.
Yeasts do not convert to spheroplast. Conditions for spheroplast formation vary according to the batch of zymolyase and/or the strains used.
Insufficient homogenization. Use the adequate volume of homogenizer or increase the number of strokes.
Binding capacity of magnetic beads may vary according to the batch. Adjust the ratio of beads and mitochondria-enriched fraction.
Mitochondria are not completely eluted. Flip tubes several times every minute.
Mitochondria are not pure Wash bead-bound mitochondria with a higher concentration of imidazole in SEH buffer or wash more than 3 times. However, high concentration of imidazole may decrease the yield.
Mitochondria are not intact Reduce the number of strokes in the homogenization steps.
Resuspend pellets more gently.
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Acknowledgements

This work was supported by grants from the National Institutes of Health (NIH) (GM45735, GM122589 and AG051047) to LP. CB, RF and FP express their gratitude to their mentor Prof. Giorgio Lenaz for having introduced them to the exciting field of mitochondrial research, for his guidance, and for his helpful suggestions

References

  1. Azzone GF, Colonna R, & Ziche B (1979). Preparation of bovine heart mitochondria in high yield. Methods Enzymol, 55, 46–50. doi: 10.1016/0076-6879(79)55007-1 [DOI] [PubMed] [Google Scholar]
  2. Banik B, Askins BW, & Dhar S (2016). Mito-magneto: a tool for nanoparticle mediated mitochondria isolation. Nanoscale, 8(47), 19581–19591. doi: 10.1039/c6nr05882e [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Banik B, & Dhar S (2017). Centrifugation-Free Magnetic Isolation of Functional Mitochondria Using Paramagnetic Iron Oxide Nanoparticles. Curr Protoc Cell Biol, 76, 25 24 21–25 24 20. doi: 10.1002/cpcb.26 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Battino M, Gorini A, Villa RF, Genova ML, Bovina C, Sassi S, … Lenaz G (1995). Coenzyme Q content in synaptic and non-synaptic mitochondria from different brain regions in the ageing rat. Mech Ageing Dev, 78(3), 173–187. doi: 10.1016/0047-6374(94)01535-t [DOI] [PubMed] [Google Scholar]
  5. Beyer RE (1967a). [34] Preparation, properties, and conditions for assay of phosphorylating electron transport particles (ETPH) and its variations. Methods Enzymol, 10, 186–194. [Google Scholar]
  6. Beyer RE (1967b). [81] The isolation, properties, and assay of ATP synthetase II. Methods Enzymol, 10, 519–522. [Google Scholar]
  7. Bharadwaj MS, Tyrrell DJ, Lyles MF, Demons JL, Rogers GW, & Molina AJ (2015). Preparation and respirometric assessment of mitochondria isolated from skeletal muscle tissue obtained by percutaneous needle biopsy. J Vis Exp(96). doi: 10.3791/52350 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Boldogh I, Vojtov N, Karmon S, & Pon LA (1998). Interaction between mitochondria and the actin cytoskeleton in budding yeast requires two integral mitochondrial outer membrane proteins, Mmm1p and Mdm10p. J Cell Biol, 141(6), 1371–1381. doi: 10.1083/jcb.141.6.1371 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Boldogh IR, & Pon LA (2007). Purification and subfractionation of mitochondria from the yeast Saccharomyces cerevisiae. Methods Cell Biol, 80, 45–64. doi: 10.1016/S0091-679X(06)80002-6 [DOI] [PubMed] [Google Scholar]
  10. Cogswell AM, Stevens RJ, & Hood DA (1993). Properties of skeletal muscle mitochondria isolated from subsarcolemmal and intermyofibrillar regions. Am J Physiol, 264(2 Pt 1), C383–389. doi: 10.1152/ajpcell.1993.264.2.C383 [DOI] [PubMed] [Google Scholar]
  11. Costantini P, Petronilli V, Colonna R, & Bernardi P (1995). On the effects of paraquat on isolated mitochondria. Evidence that paraquat causes opening of the cyclosporin A-sensitive permeability transition pore synergistically with nitric oxide. Toxicology, 99(1–2), 77–88. doi: 10.1016/0300-483x(94)02997-9 [DOI] [PubMed] [Google Scholar]
  12. Cui TZ, Conte A, Fox JL, Zara V, & Winge DR (2014). Modulation of the respiratory supercomplexes in yeast: enhanced formation of cytochrome oxidase increases the stability and abundance of respiratory supercomplexes. J Biol Chem, 289(9), 6133–6141. doi: 10.1074/jbc.M113.523688 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Daum G, Böhni PC, & Schatz G (1982). Import of proteins into mitochondria: Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. J Biol Chem, 257(21), 13028–13033. [PubMed] [Google Scholar]
  14. Devall M, Burrage J, Caswell R, Johnson M, Troakes C, Al-Sarraj S, … Lunnon K (2015). A comparison of mitochondrial DNA isolation methods in frozen post-mortem human brain tissue--applications for studies of mitochondrial genetics in brain disorders. Biotechniques, 59(4), 241–242, 244–246. doi: 10.2144/000114343 [DOI] [PubMed] [Google Scholar]
  15. Djafarzadeh S, & Jakob SM (2017). Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements. J Vis Exp(121). doi: 10.3791/55251 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Estabrook RW (1967). Mitochondrial respiratory control and polarographic measurement of ADP/O ratios. Methods Enzymol, 10, 41–47. [Google Scholar]
  17. Estornell E, Fato R, Pallotti F, & Lenaz G (1993). Assay conditions for the mitochondrial NADH:coenzyme Q oxidoreductase. FEBS Lett, 332(1–2), 127–131. doi: 10.1016/0014-5793(93)80498-j [DOI] [PubMed] [Google Scholar]
  18. Fato R, Cavazzoni M, Castelluccio C, Parenti Castelli G, Palmer G, Degli Esposti M, & Lenaz G (1993). Steady-state kinetics of ubiquinol-cytochrome c reductase in bovine heart submitochondrial particles: diffusional effects. Biochem J, 290 (Pt 1), 225–236. doi: 10.1042/bj2900225 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Franko A, Baris OR, Bergschneider E, von Toerne C, Hauck SM, Aichler M, … de Angelis MH (2013). Efficient isolation of pure and functional mitochondria from mouse tissues using automated tissue disruption and enrichment with anti-TOM22 magnetic beads. PLoS One, 8(12), e82392. doi: 10.1371/journal.pone.0082392 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Garcia-Cazarin ML, Snider NN, & Andrade FH (2011). Mitochondrial isolation from skeletal muscle. J Vis Exp(49). doi: 10.3791/2452 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Glick BS, & Pon LA (1995). Isolation of highly purified mitochondria from Saccharomyces cerevisiae. Methods Enzymol, 260, 213–223. [DOI] [PubMed] [Google Scholar]
  22. Gnaiger E (2009). Capacity of oxidative phosphorylation in human skeletal muscle: new perspectives of mitochondrial physiology. Int J Biochem Cell Biol, 41(10), 1837–1845. doi: 10.1016/j.biocel.2009.03.013 [DOI] [PubMed] [Google Scholar]
  23. Gornall AG, Bardawill CJ, & David MM (1949). Determination of serum proteins by means of the biuret reaction. J Biol Chem, 177(2), 751–766. [PubMed] [Google Scholar]
  24. Gottlieb RA, & Adachi S (2000). Nitrogen cavitation for cell disruption to obtain mitochondria from cultured cells. Methods Enzymol, 322, 213–221. doi: 10.1016/s0076-6879(00)22022-3 [DOI] [PubMed] [Google Scholar]
  25. Graham JM (2001). Isolation of mitochondria from tissues and cells by differential centrifugation. Curr Protoc Cell Biol, Chapter 3, Unit 3 3. doi: 10.1002/0471143030.cb0303s04 [DOI] [PubMed] [Google Scholar]
  26. Gregg CT (1967). [33] Preparation and assay of phosphorylating submitochondrial particles: Particles from rat liver prepared by drastic sonication. Methods Enzymol, 10, 181–185. [Google Scholar]
  27. Hansen M, & Smith AL (1964). Studies on the mechanism of oxidative phosphorylation: VII. Preparation of a submitochondrial particle (ETPH) which is capable of fully coupled oxidative phosphorylation. BBA - Enzymol. Subj, 81(2), 214–222. [Google Scholar]
  28. Hatefi Y (1978). Preparation and properties of NADH: ubiquinone oxidoreductase (complexI), EC 1.6.5.3. Methods Enzymol, 53, 11–14. doi: 10.1016/s0076-6879(78)53006-1 [DOI] [PubMed] [Google Scholar]
  29. Hornig-Do HT, Gunther G, Bust M, Lehnartz P, Bosio A, & Wiesner RJ (2009). Isolation of functional pure mitochondria by superparamagnetic microbeads. Anal Biochem, 389(1), 1–5. doi: 10.1016/j.ab.2009.02.040 [DOI] [PubMed] [Google Scholar]
  30. Hubbard WB, Harwood CL, Prajapati P, Springer JE, Saatman KE, & Sullivan PG (2019). Fractionated mitochondrial magnetic separation for isolation of synaptic mitochondria from brain tissue. Sci Rep, 9(1), 9656. doi: 10.1038/s41598-019-45568-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Khattar NK, Yablonska S, Baranov SV, Baranova OV, Kretz ES, Larkin TM, … Friedlander RM (2016). Isolation of functionally active and highly purified neuronal mitochondria from human cortex. J Neurosci Methods, 263, 1–6. doi: 10.1016/j.jneumeth.2016.01.017 [DOI] [PubMed] [Google Scholar]
  32. Lai N, C MK, Rosca MG, Fujioka H, Tandler B, & Hoppel CL (2019). Isolation of mitochondrial subpopulations from skeletal muscle: Optimizing recovery and preserving integrity. Acta Physiol (Oxf), 225(2), e13182. doi: 10.1111/apha.13182 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lee C. p., & Ernster L (1967). [87] Energy-coupling in nonphosphorylating submitochondrial particles. Methods Enzymol, 10, 543–548. [Google Scholar]
  34. Liao PC, Boldogh IR, Siegmund SE, Freyberg Z, & Pon LA (2018). Isolation of mitochondria from Saccharomyces cerevisiae using magnetic bead affinity purification. PLoS One, 13(4), e0196632. doi: 10.1371/journal.pone.0196632 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lowry OH, Rosebrough NJ, Farr AL, & Randall RJ (1951). Protein measurement with the Folin phenol reagent. J Biol Chem, 193(1), 265–275. [PubMed] [Google Scholar]
  36. Ly CV, & Verstreken P (2006). Mitochondria at the synapse. Neuroscientist, 12(4), 291–299. doi: 10.1177/1073858406287661 [DOI] [PubMed] [Google Scholar]
  37. Magalhaes PJ, Andreu AL, & Schon EA (1998). Evidence for the presence of 5S rRNA in mammalian mitochondria. Mol Biol Cell, 9(9), 2375–2382. doi: 10.1091/mbc.9.9.2375 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Marc P, Margeot A, Devaux F, Blugeon C, Corral-Debrinski M, & Jacq C (2002). Genome-wide analysis of mRNAs targeted to yeast mitochondria. EMBO Rep, 3(2), 159–164. doi: 10.1093/embo-reports/kvf025 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Meeusen S, McCaffery JM, & Nunnari J (2004). Mitochondrial fusion intermediates revealed in vitro. Science, 305(5691), 1747–1752. doi: 10.1126/science.1100612 [DOI] [PubMed] [Google Scholar]
  40. Nicholls P (1976). Catalytic activity of cytochromes c and c1 in mitochondria and submitochondrial particles. Biochim Biophys Acta, 430(1), 30–45. doi: 10.1016/0005-2728(76)90219-x [DOI] [PubMed] [Google Scholar]
  41. Pallotti F, Genova ML, Pich MM, Zucchini C, Carraro S, Tesei M, … Lenaz G (1998). Mitochondrial dysfunction and brain disorders. Archives of Gerontology and Geriatrics, 385–392. [Google Scholar]
  42. Pallotti F, & Lenaz G (2007). Isolation and subfractionation of mitochondria from animal cells and tissue culture lines. Methods Cell Biol, 80, 3–44. doi: 10.1016/S0091-679X(06)80001-4 [DOI] [PubMed] [Google Scholar]
  43. Pecinova A, Drahota Z, Nuskova H, Pecina P, & Houstek J (2011). Evaluation of basic mitochondrial functions using rat tissue homogenates. Mitochondrion, 11(5), 722–728. doi: 10.1016/j.mito.2011.05.006 [DOI] [PubMed] [Google Scholar]
  44. Rasmussen HN, Andersen AJ, & Rasmussen UF (1997). Optimization of preparation of mitochondria from 25–100 mg skeletal muscle. Anal Biochem, 252(1), 153–159. doi: 10.1006/abio.1997.2304 [DOI] [PubMed] [Google Scholar]
  45. Reijnierse GL, Veldstra H, & Van den Berg CJ (1975). Subcellular localization of gamma-aminobutyrate transaminase and glutamate dehydrogenase in adult rat brain. Evidence for at least two small glutamate compartments in brain. Biochem J, 152(3), 469–475. doi: 10.1042/bj1520469 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Reinders J, Wagner K, Zahedi RP, Stojanovski D, Eyrich B, van der Laan M, … Meisinger C (2007). Profiling phosphoproteins of yeast mitochondria reveals a role of phosphorylation in assembly of the ATP synthase. Mol Cell Proteomics, 6(11), 1896–1906. doi: 10.1074/mcp.M700098-MCP200 [DOI] [PubMed] [Google Scholar]
  47. Reinhart PH, Taylor WM, & Bygrave FL (1982). A procedure for the rapid preparation of mitochondria from rat liver. Biochem J, 204(3), 731–735. doi: 10.1042/bj2040731 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Rieske JS (1967). [44] Preparation and properties of reduced coenzyme Q-cytochrome c reductase (complex III of the respiratory chain). Methods Enzymol, 10, 239–245. [Google Scholar]
  49. Schmitt S, Eberhagen C, Weber S, Aichler M, & Zischka H (2015). Isolation of mitochondria from cultured cells and liver tissue biopsies for molecular and biochemical analyses. Methods Mol Biol, 1295, 87–97. doi: 10.1007/978-1-4939-2550-6_8 [DOI] [PubMed] [Google Scholar]
  50. Schneiter R, Brugger B, Sandhoff R, Zellnig G, Leber A, Lampl M, … Kohlwein SD (1999). Electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of the lipid molecular species composition of yeast subcellular membranes reveals acyl chain-based sorting/remodeling of distinct molecular species en route to the plasma membrane. J Cell Biol, 146(4), 741–754. doi: 10.1083/jcb.146.4.741 [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Simpson RJ (2010). Disruption of cultured cells by nitrogen cavitation. Cold Spring Harb Protoc, 2010(11), pdb prot5513. doi: 10.1101/pdb.prot5513 [DOI] [PubMed] [Google Scholar]
  52. Smith AL (1967). [13] Preparation, properties, and conditions for assay of mitochondria: Slaughterhouse material, small-scale. Methods Enzymol, 10, 81–86. [Google Scholar]
  53. Stuart RA, & Koehler CM (2007). In vitro analysis of yeast mitochondrial protein import. Curr Protoc Cell Biol, Chapter 11, Unit 11 19. doi: 10.1002/0471143030.cb1119s34 [DOI] [PubMed] [Google Scholar]
  54. Tapper DP, Van Etten RA, & Clayton DA (1983). Isolation of mammalian mitochondrial DNA and RNA and cloning of the mitochondrial genome. Methods Enzymol, 97, 426–434. doi: 10.1016/0076-6879(83)97153-7 [DOI] [PubMed] [Google Scholar]
  55. Van den Berg CJ (1973). A Model of Compartmentation in Mouse Brain Based on Glucose and Acetate Metabolism. In Balázs R & Cremer JE (Eds.), Metabolic Compartmentation in the Brain (pp. 137–166). London: Palgrave. [Google Scholar]
  56. Vanneste WH (1966). Molecular proportion of the fixed cytochrome components of the respiratory chain of Keilin-Hartree particles and beef heart mitochondria. Biochim Biophys Acta, 113(1), 175–178. doi: 10.1016/s0926-6593(66)80132-7 [DOI] [PubMed] [Google Scholar]
  57. Vos M, Lauwers E, & Verstreken P (2010). Synaptic mitochondria in synaptic transmission and organization of vesicle pools in health and disease. Front Synaptic Neurosci, 2, 139. doi: 10.3389/fnsyn.2010.00139 [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wang WX, Sullivan PG, & Springer JE (2017). Mitochondria and microRNA crosstalk in traumatic brain injury. Prog Neuropsychopharmacol Biol Psychiatry, 73, 104–108. doi: 10.1016/j.pnpbp.2016.02.011 [DOI] [PubMed] [Google Scholar]
  59. Wang X, Leverin AL, Han W, Zhu C, Johansson BR, Jacotot E, … Hagberg H (2011). Isolation of brain mitochondria from neonatal mice. J Neurochem, 119(6), 1253–1261. doi: 10.1111/j.1471-4159.2011.07525.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wettmarshausen J, & Perocchi F (2017). Isolation of Functional Mitochondria from Cultured Cells and Mouse Tissues. Methods Mol Biol, 1567, 15–32. doi: 10.1007/978-1-4939-6824-4_2 [DOI] [PubMed] [Google Scholar]
  61. Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, … Wang X (1997). Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science, 275(5303), 1129–1132. doi: 10.1126/science.275.5303.1129 [DOI] [PubMed] [Google Scholar]

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