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Organelle isolation: functional mitochondria from mouse liver, muscle and cultured filroblasts

Nature Protocols

Volume 2 Issue 2, , Pages 287 - 295

doi: 10.1038/nprot.2006.478

Nature Publishing Group


, ,

a Dulbecco-Telethon Institute, Venetian Institute of Molecular Medicine


Abstract

Mitochondria participate in key metabolic reactions of the cell and regulate crucial signaling pathways including apoptosis. Although several approaches are available to study mitochondrial function in situ are available, investigating functional mitochondria that have been isolated from different tissues and from cultured cells offers still more unmatched advantages. This protocol illustrates a step-by-step procedure to obtain functional mitochondria with high yield from cells grown in culture, liver and muscle. The isolation procedures described here require 1–2 hours, depending on the source of the organelles. The polarographic analysis can be completed in 1 hour.


Materials

Reagents
  • Cell line of interest or liver or muscle isolated from mice

  • Mice of the desired genetic background (Charles River or Jackson Laboratories)

  • Dulbecco's phosphate-buffered saline without Ca2+ and Mg2+ (PBS, Invitrogen, cat. no. 14200-067)

  • Sucrose (Sigma, cat. no. 84100)

  • Potassium phosphate monobasic (Pi, Sigma, cat. no. P5379)

  • Sigma7-9 (Tris, Sigma, cat. no. T1378)

  • 4-Morpholinepropanesulfonic acid (MOPS; Sigma, cat. no. M1254)

  • Disodium ethylenediaminetetraacetate dihydrate (EDTA; Sigma, cat. no. ED2SS)

  • Ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA; Sigma, cat. no. E4378)

  • Potassium chloride (Baker, cat. no. 0208)

  • Magnesium chloride hexahydrate (Sigma, cat. no. M9272)

  • Bovine serum albumin (BSA; Sigma, cat. no. A6003)

  • Dulbecco's modified Eagle's medium (Invitrogen, cat. no. 11971025)

  • 200 mM L-glutamine (Invitrogen, cat. no. 25030024),

  • Fetal bovine serum (Invitrogen, cat. no. 10270106)

  • 5,000 U ml−1 penicillin/5,000 μg ml−1 streptomycin (Invitrogen, cat. no. 15070063)

  • 10 mM minimal essential medium nonessential amino-acid solution (Invitrogen, cat. no. 11140)

  • 0.25% (w/v) trypsin–EDTA solution (Invitrogen, cat. no. 25200072)

  • Adenosine 5′-diphosphate sodium salt (ADP; Sigma, cat. no. A2754)

  • Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; Sigma, cat. no. C2920)

  • Glutamic acid (Sigma, cat. no. 27647)

  • Malic acid (Sigma, cat. no. M1000)

  • Succinic acid (Sigma, cat. no. S3674)

  • Rotenone (Sigma, cat. no. R8875)

  • L-Ascorbic acid (Sigma, cat. no. 255564)

  • N,N,N,N-Tetramethyl-p-phenylenediamine dihydrochloride (TMPD; Sigma, cat. no. T3134)

  • Antimycin A (Sigma, cat. no. A8674)

Equipment
  • 500 cm2 dishes for cell culture (Nunclon, cat. no. 16 6508)

  • 18-cm cell scrapers (Falcon, cat. no. 353085)

  • Motor-driven tightly fitting glass/Teflon Potter Elvehjem homogenizer (Fig. 1)

  • Clark-type oxygen electrode (Hansatech Oxygraph; Fig. 2)

  • 50 ml polypropylene Falcon tubes

  • 14 ml polypropylene Falcon tubes

  • 1.5 ml microfuge test tube

  • 30 ml round-bottomed glass centrifuge tube (Kimble, cat. no. 45500-30)

  • Rubber adapter sleeve for centrifuge tube (Kimble, cat. no. 45500-15)

  • Refrigerated centrifuge for 50 ml Falcon tubes and glass centrifuge tube

  • Hamilton syringe: 10 μl (Hamilton, cat. no. 701 N) and 50 μl (Hamilton cat. no. 705 N)

Reagent setup
  • Cell culture medium Use the medium recommended for your favorite cell line. For the cell lines mentioned in this protocol, use Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 0.1 mM minimal essential medium nonessential amino acids, 2 mM L-glutamine, penicillin–streptomycin 50 U ml−1 and 50 μg ml−1, respectively.

  • Cells Two or three days before performing the experiments, plate cells in 500 cm2 tissue-culture dishes. Use 70 ml of cell culture medium for each plate.

    • Ensure that the cells are spread thoroughly wide on the plates: for high yield of isolated mitochondria, it is crucial to reach almost 100% confluence on the day of the experiment.

  • 1 M sucrose Dissolve 342.3 g of sucrose in 1 liter of distilled water; mix well and prepare 20 ml aliquots; store them at −20 °C.

  • 0.1 M Tris/MOPS Dissolve 12.1 g of Tris in 500 ml of distilled water, adjust pH to 7.4 using MOPS powder, bring the solution to 1 liter and store at 4 °C.

  • 1 M Tris/HCl Dissolve 121.14 g of Tris in 500 ml of distilled water, adjust pH to 7.4 using HCl; bring the solution to 1 liter and store at room temperature.

  • 0.1 M EGTA/Tris Dissolve 38.1 g of EGTA in 500 ml of distilled water, adjust pH to 7.4 using Tris powder, bring the solution to 1 liter and store at 4 °C.

  • 0.5 M MgCl2 Dissolve 101.7 g of MgCl2 in 1 liter of distilled water and store at 4 °C.

  • 1 M KCl Dissolve 74.6 g of KCl in 1 liter of distilled water and store at 4 °C.

  • 1 M EDTA Dissolve 372.2 g of EDTA in 500 ml of distilled water, adjust pH to 7.4 using Tris powder, bring the solution to 1 liter and store at 4 °C.

  • 10% BSA Dissolve 10 g of BSA in 100 ml of distilled water and store at −20 °C.

  • 1 M Pi Dissolve 136.1 g of KH2PO4 in 500 ml of distilled water, adjust pH to 7.4 using Tris powder, bring the solution to 1 liter and store at 4 °C.

  • 10 mM ADP Dissolve 4.7 mg of ADP in 1 ml of distilled water. Adjust pH to 7.4, prepare 100 μl aliquots and store in the dark at −20 °C for up to 6 months.

  • 20 mM FCCP Dissolve 5.1 mg of FCCP in 1 ml of absolute ethanol. The color of the solution is faint yellow. Store at −20 °C. Dilute the stock solution to 100 μM by adding 10μl of 20 mM FCCP in 2 ml of absolute ethanol, just prior to use.

  • 0.25 M glutamate/0.125 M malate Dissolve 9.2 g of glutamic acid and 4.2 g of malic acid in 100 ml of distilled water. Adjust pH to 7.4 with Tris base to achieve complete dissolution of the salts. Add water to bring the volume to 250 ml, prepare 10 ml aliquots and store at −20 °C for up to 6 months.

  • 0.5 M succinate stock solution (100 ×) Dissolve 3.0 g of succinic acid in 30 ml of distilled water. Adjust pH with Tris base to achieve complete solubilization of the salts. Add water to make up the volume to 50 ml, prepare 10 ml aliquots and store at −20 °C for up to 6 months.

  • 2 mM rotenone stock solution Dissolve 4.7 mg of rotenone in 6 ml of absolute ethanol. Mix well for complete dissolution.

    • Rotenone in organic solvents decomposes and is oxidized upon exposure to light and air. The solution, previously transparent, becomes brownish. It is imperative to protect the stock solution from direct light using an aluminum foil.

    Rotenone is highly toxic: avoid skin contact and inhalation.

  • 600 mM ascorbate stock solution Dissolve 5.2 g of ascorbic acid in 50 ml of distilled water, adjust pH to 7.4 and store at −20 °C for up to 6 months.

  • 30 mM TMPD stock solution Dissolve 0.36 g of TMPD in 50 ml of distilled water; adjust pH to 7.4; store at −20 °C for up to 6 months. The color of the solution is deep blue owing to the oxidation of the compound by oxygen.

  • 25 mg ml−1 antimycin A stock solution Dissolve 50 mg of antimycin A in 2 ml of absolute ethanol. Dilute the stock solution to 25 μg ml−1, by adding 2 μl of 25 mg ml−1 Antimycin A in 2 ml of absolute ethonal, just prior to use.

    Antimycin A is highly toxic: avoid skin contact and inhalation.

  • Buffer for cell and mouse liver mitochondria isolation (IBc) Prepare 100 ml of IB by adding 10 ml of 0.1 M Tris–MOPS and 1 ml of EGTA/Tris to 20 ml of 1 M sucrose. Bring the volume to 100 ml with distilled water. Adjust pH to 7.4.

  • Buffer 1 for muscle mitochondria isolation (IBm1) Prepare 100 ml of IBm1 by mixing 6.7 ml of 1 M sucrose, 5 ml of 1 M Tris/HCl, 5 ml of 1 M KCl, 1 ml of 1 M EDTA and 2 ml of 10% BSA. Adjust pH to 7.4. Bring the volume to 100 ml with distilled water.

  • Buffer 2 for muscle mitochondria isolation (IBm2) Prepare 100 ml of IBm12 by mixing 25 ml of 1 M sucrose, 3 ml of 0.1 M EGTA/Tris and 1 ml of 1 M Tris/HCl. Adjust pH to 7.4. Bring the volume to 100 ml with distilled water.

  • Experimental buffer for cell and mouse-liver mitochondria (EBc) To prepare 100 ml of EBc, mix 12.5 ml of 1 M KCl, 1 ml of 1 M Tris/MOPS, 10 ml of 100 μl 0.1 M EGTA/Tris and 100 μl of Pi. Adjust pH to 7.4. Bring the volume to 100 ml with distilled water.

  • Experimental buffer for muscle mitochondria (EBm) To prepare 100 ml of EBm, add 1 ml of 1 M Tris/HCl, 1 ml of 0.5 M MgCl2, 200 μl of 1 M Pi and 20 μl of 0.1 M EGTA/Tris to 25 ml of 1 M sucrose. Adjust pH to 7.4. Bring the volume to 100 ml with distilled water.

    • Wash all glassware three times with bidistilled water to avoid Ca2+ contamination. Ca2+ overload is the most common cause for the dysfunction of isolated mitochondria.

    • Prepare all the buffers the same day of the experiment, to avoid bacterial/yeast growth in stored buffers.

    • Since pH depends on temperature, measure the pH of all solutions at 25 °C.


Materials List

     

Procedure

  • Mitochondria can be isolated from a variety of cells or tissues. Option A describes isolation of mitochondria from mouse embryonic fibroblasts (MEFs) (see Fig. 3 for a timeline); option B describes isolation of mitochondria from mouse liver (see Fig. 4 for a timeline); and option C describes isolation of mitochondria from mouse skeletal muscle (see Fig. 5 for a timeline).

    • Isolation of mitochondria from MEFs

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

      • Remove PBS and detach the cells using a cell scraper.

      • Transfer the cell suspension to a 50 ml polypropylene Falcon tube.

      • Wash the plate once with PBS and scrape the dish to detach the remaining cells.

      • Transfer the cells to the same polypropylene Falcon tube defined in Step 3. In our experience, seeding 120 × 106 MEFs per dish 2 days before the experiment results in a good yield of mitochondria (approximately 3 mg of mitochondrial protein).

      • Centrifuge cells at 600g at 4 °C for 10 min.

      • Discard the supernatant and resuspend cells in 3 ml of ice-cold IBc.

      • Homogenize the cells using a Teflon pestle operated at 1,600 r.p.m.; stroke the cell suspension placed in a glass potter 30–40 times the cell suspension placed in a glass potter.

        • The Teflon–glass coupling represents the best compromise between homogenization of the cells and the preservation of mitochondrial integrity. Harsher techniques, including glass pestle in a glass potter, can easily damage mitochondria.

        • Precool the glassware in an ice-bath 5 min before starting the procedure. Homogenization as well as the following steps must be performed at 4 °C to minimize the activation of damaging phospholipases and proteases.

        Wear protecting gloves while you are using the homogenizer to avoid possible injuries in the unlikely event that the potter breaks down.

      • Transfer the homogenate to a 50 ml polypropylene Falcon tube and centrifuge at 600g for 10 min at 4 °C.

      • Collect the supernatant, transfer it to a glass centrifuge tube and centrifuge it at 7,000g for 10 min at 4 °C.

      • Discard the supernatant and wash the pellet with 200 μl of ice-cold IBc. Resuspend the pellet in 200 μl of ice-cold IBc and transfer the suspension to a 1.5 ml microfuge tube.

      • Centrifuge the homogenate at 7,000g for 10 min at 4 °C.

      • Discard the supernatant and resuspend the pellet containing mitochondria. You can use a glass rod to loosen the pellet paste. Avoid adding IB and try to resuspend the mitochondria in the small amount of buffer that remains after discarding the supernatant. Use a 200 μl pipettor and avoid the formation of bubbles during the resuspension.

      • Transfer the mitochondrial suspension to a microfuge and store it on ice.

        • Avoid diluting mitochondria with buffer. Mitochondria retain their functionality for a longer time, probably as a consequence of lower exposure to oxygen, when they are stored in a concentrated form.

      • Measure mitochondria concentration using the Biuret methods.

        • Mitochondria are now ready to be used in experiments: use the preparation within 1–3 h for better functional responses.

        • The typical yield of this preparation is ∼50 mg ml−1 in a total volume of approximately 0.1 ml.

        • The Biuret method for measurement of mitochondrial concentration is accurate in the range of protein concentrations obtained from this protocol; other methods like the Bradford method can be used, but the mitochondrial lysate must be diluted in order to avoid saturation of the probe.

      approximately 2 h

    • Isolation of mitochondria from mouse liver

      • Starve the mouse overnight before the isolation experiment.

      • Kill an adult mouse (about 30 g) by cervical dislocation and rapidly explant the liver from the peritoneal cavity. Find the gallbladder and remove it using a scalpel. Immerse the liver in 50 ml of ice-cold IBc in a small beaker.

        • Local and national regulations on animal care and handling vary. Check that you hold the appropriate authorization to perform animal experiments.

      • Rinse the liver free of blood by using ice-cold IBc. Usually, four or five washes are sufficient to completely clarify the IBc.

      • Mince the liver into small pieces using scissors. This should be performed while keeping the beaker in an ice bath.

      • Discard the IBc used during the mincing and replace it with 5 ml of ice-cold fresh IBc. Transfer the suspension to the glass potter.

        • Homogenization, as well as the following steps, must be performed at 4 °C to minimize activation of damaging phospholipases and proteases.

      • Homogenize the liver using a Teflon pestle operated at 1,600 r.p.m., stroke the minced liver 3–4 times.

        • The optimal ratio between tissue and isolation buffer ranges between 1:5 to 1:10 (w:v).

        • Precool the glassware in an ice-bath 5 min before starting the procedure. Homogenization and the following steps must be performed at 4 °C to minimize activation of damaging phospholipases and proteases.

        Wear protecting gloves while you are using the homogenizer to avoid possible injuries in the unlikely event that the potter breaks down.

      • Transfer the homogenate to a 50 ml polypropylene Falcon tube and centrifuge at 600g for 10 min at 4 °C.

      • Transfer the supernatant to glass centrifuge tubes and centrifuge at 7,000g for 10 min at 4 °C.

      • Discard the supernatant and wash the pellet with 5 ml of ice-cold IBc.

      • Centrifuge at 7,000g for 10 min at 4 °C.

      • Discard the supernatant and resuspend the pellet, containing mitochondria. You can use a glass rod to loosen the pellet paste. Avoid adding IB and try to resuspend the mitochondria in the small amount of buffer that remains after discarding the supernatant. Use a 1 ml pipettor and avoid the formation of bubbles during the resuspension process.

      • Transfer mitochondrial suspension into a 14 ml Falcon tube and store on ice.

        • Avoid diluting mitochondria with buffer as mitochondria retain their functionality for a longer time when kept concentrated, minimizing exposure to oxygen.

        • Mitochondria are now ready to be used in experiments; use the preparation within 1–3 h for better functional responses.

      • Measure mitochondrial concentration using the Biuret methods.

        • The usual concentration of mitochondria in this kind of preparation is about 80 mg ml−1 and the total volume is about 1 ml.

        • The Biuret method for measurement of mitochondrial concentration is accurate in the range of protein concentrations obtained from this protocol; other methods like the Bradford method can be used, but the mitochondrial lysate must be diluted in order to avoid saturation of the probe.

      approximately 1 h

    • Isolation of mitochondria from mouse skeletal muscle

      • Kill the mouse by cervical dislocation. Using a scalpel, rapidly remove the skeletal muscles of interest and immerse them in a small beaker containing 5 ml of ice-cold PBS supplemented with 10 mM EDTA. A timeline of this protocol is outlined in Figure 6.

        • Local and national regulations on animal care and handling vary. Check that you hold the appropriate authorizations to perform animal experiments.

        • The use of EDTA instead of EGTA chelates also Mg2+, which is extremely abundant in muscle tissue (given the high content in ATP). Mg2+ can influence mitochondrial function as well as the kinetics of cytochrome c release 25 .

      • Mince the muscles into small pieces using scissors and trim visible fat, ligaments and connective tissue.

      • Wash the minced muscles twice or thrice with ice-cold PBS supplemented with 10 mM EDTA.

      • Resuspend the minced muscles in 5 ml of ice-cold PBS supplemented with 10 mM EDTA and 0.05% trypsin for 30 min.

      • Centrifuge at 200g for 5 min and discard the supernatant.

      • Resuspend the pellet in IBm1.

      • Homogenize the muscles using a Teflon pestle operated at 1,600 r.p.m.; stroke the minced muscle ten times.

        • The optimal ratio between tissue and isolation buffer ranges between 1:5 and 1:10 (w:v).

        • Precool the glassware in an ice-bath 5 min before starting the procedure. Homogenization, and the following steps, must be performed at 4 °C to minimize the activation of damaging phospholipases and proteases.

        Wear protecting gloves while you are using the homogenizer to avoid possible injuries in the unlikely event that the potter breaks down.

        Precool the glassware in an ice-bath for 5 min before starting the following steps.

      • Transfer the homogenate to a 50 ml polypropylene Falcon tube and centrifuge at 700g for 10 min at 4 °C.

      • Transfer the supernatant to glass centrifuge tubes and centrifuge at 8,000g for 10 min at 4 °C.

      • Discard the supernatant and resuspend the pellet in 5 ml of ice-cold IBm2.

      • Centrifuge at 8,000g for 10 min at 4 °C.

      • Discard the supernatant and resuspend the pellet containing mitochondria. You can use a glass rod to loosen the pellet paste. Avoid adding IB and try to resuspend the mitochondria in the small amount of buffer that remains after discarding the supernatant. Use a 200 ml pipettor and avoid the formation of bubbles during the resuspension process.

      • Transfer mitochondrial suspension into a 14 ml Falcon tube and keep it on ice.

      • Measure mitochondrial concentration using the Biuret methods.

        • This preparation normally yields 0.8 ml of 50 mg ml−1 mitochondria.

        • The Biuret method for measurement of mitochondrial concentration is accurate in the range of protein concentrations obtained from this protocol; other methods like the Bradford method can be used, but the mitochondrial lysate must be diluted in order to avoid saturation of the probe.

      approximately 1.5 h

Measuring mitochondrial respiration

approximately 1 h

  • Calibrate the Clarke-type oxygen electrode. Procedures vary from instrument to instrument. You should follow the manufacturer's instructions for the instrument you are using.

  • Equilibrate temperature and oxygen tension of EBc or EBm by placing open beakers containing the buffers in the water bath connected to the oxygraph. After 20–30 min, the temperature of the buffers is likely to be in equilibrium with that of the water bath.

  • Add an appropriate volume of EB to the oxygraph chamber. Use 0.5 ml for the mitochondria isolated from cells and 1 or 2 ml for the liver and muscle mitochondria. Close the oxygraph chamber.

  • Start the recording of the oxygen consumption.

    • Verify that the recording is stable and that no drifts are apparent. Drifts can mask the oxygen consumption by the mitochondrial preparation and thereby complicate the interpretation of the results.

  • Wait for 2 min to obtain a stable baseline.

  • Using an appropriate Hamilton microsyringe, add mitochondria to obtain a final concentration of 1 mg ml−1. A fast, transitory decrease in the oxygen content of the chamber will be observed, caused by anaerobiosis of the isolated mitochondria; this will be followed by a slower decrease caused by the respiration of the mitochondria. This is supported by endogenous substrates and is commonly referred to as “state 1” respiration 26 .

  • Record oxygen consumption till it stops.

    In liver mitochondria state 1 respiration commonly does not stop.

  • Using Hamilton microsyringes, add the appropriate concentrations of respiratory substrates and inhibitors for the complexes of the respiratory chain you wish to study (refer to Table 1). The mitochondrial suspension will now start consuming oxygen as a consequence of the basal activity of the respiratory chain in counteracting the inner mitochondrial membrane proton leak. This represents the so-called “state 2” respiration 26 .

    • The rate of oxygen consumption should now be faster than the rate observed with buffer alone. This indicates that you have obtained functional, respiring mitochondria.

  • Record for 5 min.

  • Add ADP to obtain a final concentration of 100–150 μM. Faster consumption of oxygen will be observed. This has been caused by proton back-diffusion through the stalk portion of the ATPase, which has been compensated by faster electron flow through the respiratory chain to the terminal electron acceptor, O2. This is classically referred to as “state 3” respiration 26 .

    • The rate of oxygen consumption should now be faster than the rate observed with substrates alone, indicating that we have obtained well coupled mitochondria. The increase in respiration, observed with ADP, varies from tissue to tissue and from substrate to substrate. As a general rule, and for the sole purpose of quality control of the preparation, the minimum requirements to proceed with the experiment are as follows: using glutamate malate as a substrate, maintaining a ratio of 2 in mitochondria isolated from cell lines and a ratio of 4 in mitochondria isolated from tissues.

  • Wait until the respiration slows down and returns to a rate comparable to that before the addition of ADP. This is caused by the consumption of the added ADP. The respiration, which follows ADP exhaustion, is classically referred to as “state 4” respiration 26 .

  • Wait for 3 min.

  • Add the uncoupler FCCP to obtain a final concentration of 60–100 nM.

  • The respiration will speed up and reach values slightly higher than those observed during the recording of state-3 respiration.

  • Record for a further 5 min and then stop recording.


Acknowledgements

We thank Giuliano Dodoni and members of the Scorrano lab for helpful discussions. LS is an Assistant Telethon Scientist of the Dulbecco-Telethon Institute. Research in his laboratory is supported by Telethon Italy; AIRC Italy; Compagnia di San Paolo; Human Frontier Science Program Organization; United Mitochondrial Disease Fund USA, Muscular Dystrophy Association USA.


References

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Figures

Glass/Teflon Potter Elvehjem homogenizers.

Glass/Teflon Potter Elvehjem homogenizers.

Glass/Teflon Potter Elvehjem homogenizers.

The homogenizer on the left (5 ml) is most suitable for isolation of mitochondria from cells, whereas the one on the right (30 ml) is more appropriate for isolation from tissues.

A Clarke-type oxygen electrode connected to a laptop and a water bath.

A Clarke-type oxygen electrode connected to a laptop and a water bath.

A Clarke-type oxygen electrode connected to a laptop and a water bath.

The trace on the screen corresponds to the recording of the experiment running when the photograph was taken.

Timing of isolation of mitochondria from MEFs.

Timing of isolation of mitochondria from MEFs.

Timing of isolation of mitochondria from MEFs.

Timing of isolation of mitochondria from mouse liver.

Timing of isolation of mitochondria from mouse liver.

Timing of isolation of mitochondria from mouse liver.

Timing of isolation of mitochondria from mouse skeletal muscle.

Timing of isolation of mitochondria from mouse skeletal muscle.

Timing of isolation of mitochondria from mouse skeletal muscle.

Ultrastructure and oxygen consumption of mouse liver mitochondria isolated according to the protocol presented.

Ultrastructure and oxygen consumption of mouse liver mitochondria isolated according to the protocol presented.

Ultrastructure and oxygen consumption of mouse liver mitochondria isolated according to the protocol presented.

(a) Mitochondrial ultrastructure. Mouse liver mitochondria (0.5 mg ml−1) incubated in EB supplemented with 5 mM glutamate and 2.5 mM malate for 5 min were fixed by adding glutaraldehyde (final concentration 2.5% (v/v)). Transmission electron micrographs were acquired from randomly selected fields, as described 18 . (b) Oxygen consumption of 1 ml EB supplemented with 5 mM glutamate and 2.5 mM malate. Where indicated (arrows), mouse liver mitochondria (MLM, final concentration 1 mg ml−1), ADP (100 μM) and FCCP (60 nM) were added. Respiration after ADP stimulation is indicated as “state 3”, whereas respiration after consumption of added ADP is indicated as “state 4.”


Tables

Table 1

Substrates and inhibitors of the respiratory chain.
Substrate (final concentration) Inhibitor (final concentration)
Complexes I, III, IV Glutamate (5 mM)/malate (2.5 mM) ///
Complexes II, III, IV Succinate (5 mM) Rotenone (2 μM)
Complex IV Ascorbate (6 mM)/TMPD (300 μM) Antimycin A (0.25 μg ml−1)

The span of the respiratory chain examined by each combination of substrate/inhibitor is indicated along with the final concentration to use in the oxygraphy experiments.

Table 2

Troubleshooting table.
Step Problem Possible causes Solution
1Aviii, 1Bvi, 1Cvii Low yield of isolated mitochondria Low cell density during homogenization Low cell density may result in better homogenization. However, mitochondria are usually of lower quality, probably as a consequence of mechanical damage during preparation
1Ax, 1Bviii, 1Cix Low quality of isolated mitochondria Pellet after centrifugation is lost When the supernatant is poured off, the loose upper part of the mitochondrial pellet may be detached as well. Intact mitochondria tend to sediment more quickly than damaged mitochondria. The loose part of the pellet probably contains a high proportion of damaged (uncoupled) mitochondria and can be lost without affecting the overall quality of the mitochondrial preparation
1Ax, 1Bvii Low quality of isolated mitochondria Lipid contamination The white foamy material near the top of the tube consists of lipids. Mixing of lipids with the mitochondria suspension will cause some degree of uncoupling. Therefore avoid contact with mitochondria: remove the foamy material by wiping the inside of the tube with a Kimwipe
5 Oxygen consumption baseline is not stable Bacterial or yeast contamination of your buffer Verify if your buffers are contaminated, by repeating the recording with bidistilled water in the oxygraph chamber
Inadequate calibration of the instrument Re-calibrate the instrument
Tears in the polyethylene membrane of the electrode Check response of the oxygraph by transiently stopping stirring: due to the immediate drop in the local oxygen concentration the recording should immediately fall and return to the original baseline only when the stirrer is restarted. If this manoeuvre does not give the expected results, inspect and if necessary substitute the membrane of the electrode
9 Mitochondrial preparation is not consuming oxygen Overestimation of final protein concentration (therefore added too little protein in the oxygraph chamber) Try to double mitochondrial concentration in the chamber
Mechanical and osmotical damage to mitochondria during isolation Substitute 0.2 M sucrose with 0.3 M mannitol in the isolation buffer
Contamination by other intracellular membranes, such as endoplasmic reticulum or nuclei In steps 1Axi, 1Bix or 1Cx, wash the mitochondrial pellet with twice the amount of isolation buffer
11 ADP-stimulated respiration rate is too low The most trivial explanation is that you omitted Pi from your buffer Add Pi and check the respiration
High percentage of mitochondria with ruptured outer membranes that leaked cytochrome c Add exogenous cytochrome c and check the respiration; if respiration starts, the outer membrane is leaky. See troubleshooting for step 9 (mechanical and osmotic damage)
Unusually high basal respiration, as a consequence of uncoupling by Ca2+ overload Follow carefully all the indicated critical steps to avoid the indicated contaminations; try washing glassware with isolation buffer, supplemented with EGTA
Unusually high basal respiration, as a consequence of uncoupling by fatty acids Include 0.1% fatty acid free albumin in the EB; if this procedure works increase FCCP concentration since albumin binds reversibly to FCCP
15 Uncoupled respiration rate is lower than ADP-stimulated respiration Too much FCCP used Since at high doses FCCP is also an inhibitor of the respiratory chain, you can overcome this problem by titrating down the concentration of FCCP used

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