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.
Mitochondria are central organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP and regulate a number of signaling cascades, including apoptosis1.
Since the early years of “hard-core” bioenergetics when mechanisms behind energy conservation were avidly investigated, mitochondrial research has benefited from the availability of preparations of organelles isolated from tissues. We owe this to the pioneering work of George Palade and coworkers, who in the late 1940s developed a protocol to isolate mitochondria, based on differential centrifugation2. They built on the earlier work of Bensley and Hoerr3, who isolated a mixed membranous fraction by centrifugation from freeze-thawed guinea-pig liver that was probably enriched in mitochondria. The intuition of Palade was to apply differential centrifugation to allow for separation of the constituents of the cell based on their different sedimentation properties following mechanical homogenization of the tissue. This approach was a real Copernican revolution for mitochondrial research, allowing the isolation of pure organelles with high yields. As a practical consequence, in the subsequent 20 years, we saw such amazing discoveries: the mechanism of energy conservation4; the identification of mitochondrial DNA5,6 and of import of mitochondrial precursor proteins7; the definition of mitochondrial ultrastructure, with the development of the so-called “Palade's model”8; and last but not least, the discovery of inner mitochondrial membrane channels9.
After almost 15 years during which mitochondria left the center stage of biomedical research, they made their grand reentrée in the 1990s, following the discovery that they amplify apoptosis by releasing cytochrome c and other intermembrane space proteins required to activate fully effector caspases10,11. Although it appears clear that mitochondria play a crucial role in apoptosis, the precise mechanism by which cytochrome c is released remains a matter of intense debate and research12. Moreover, evidence is mounting on the role of this organelle in several pathophysiological processes, including neurodegeneration13, neuronal morphogenesis and plasticity14 and infertility15. These findings, added to the results of old and new areas of research, aimed at unraveling the basic biological mechanisms of mitochondrial function. From the transport of metabolites and ions, to the elucidation of the mechanisms and proteins involved in protein import, and to the dynamic behavior of mitochondria, all of these fields benefit greatly from the availability of isolated, pure organelles.
This protocol describes how to obtain functional, purified, intact mitochondria from three different sources: liver16, skeletal muscle17 and cultured cells18. These variants intend to be exemplificative and not exhaustive, as they do not cover the different sources from which mitochondria can be isolated. For example, isolation of mitochondria from yeast cells is tailored on the mechanical and osmotic characteristics of these lower eucaryotes19. Since our intention is to give a general framework for different organs and for cultured cells that can be in any case modified by the individual researcher, following exactly these protocols is best suited only for isolation of organelles from the described tissues and cells. However, our experience indicates that the protocol used with fibroblasts can be adopted without modification to isolate mitochondria from other cell lines such as HeLa and the prostate cancer cell line LnCaP. On the other hand, the protocols to isolate mitochondria from organs other than muscle and liver differ from the ones described here. We therefore strongly advise the reader to refer to published protocols specific for brain20, brown adipose tissue21, and heart22.
It should be stressed that protocols available to isolate mitochondria are somewhat differ from ours, especially in the speeds of the differential centrifugation steps and in the sugar used as osmolyte in the isolation buffer. While in our experience small changes in the sedimentation speeds (600 vs. 800g, 7,000 vs. 8,000g) do not affect quality and yield of the mitochondrial preparation, it has been reported that the use of monosaccharides such as mannitol results in better coupled isolated mitochondria23,24. In our experience the use of mannitol did not improve the quality of our mitochondrial preparations. Should the reader find that quality or yield of mitochondria isolated using our protocol is unsatisfactory, it is advisable to try to substitute sucrose with a monosaccharide like mannitol. The ultimate goal of a mitochondrial isolation is to obtain organelles as pure and as functional as possible. We strongly advise, especially if mitochondria are used in functional assays (e.g., release of cytochrome c, mitochondrial fusion, protein import and production of reactive oxygen species), to always measure the coupling of the preparation using an oxygen electrode. These protocols therefore end with a description of how to measure mitochondrial respiration to ascertain the quality of the preparation. Well-coupled mitochondria are the first step to achieving reliable, reproducible results in assays aimed at investigating the mechanisms of mitochondrial involvement in complex biological phenomena.
In conclusion, these protocols represent a valuable starting point to obtain pure mitochondria from tissues and cells. Isolated mitochondria can then be used to study the function of the organelle, response to apoptotic stimuli, characteristics of cytochrome c release, protein import and many other aspects of mitochondrial biology and pathophysiology that require a source of pure and functional organelles.
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)
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)
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.
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.
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.
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.
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.
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.
Measure mitochondria concentration using the Biuret methods.
Timing: 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.
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.
Homogenize the liver using a Teflon pestle operated at 1,600 r.p.m., stroke the minced liver 3–4 times.
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.
Measure mitochondrial concentration using the Biuret methods.
Timing: 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.
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.
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.
Timing: approximately 1.5 h
Measuring mitochondrial respiration
Timing: 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.
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” respiration26.
Record oxygen consumption till it stops.
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” respiration26.
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” respiration26.
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” respiration26.
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.
Troubleshooting advice can be found in Table 2.
Step 1A: approximately 2 h, depending on the amount of cells to be used; however, cells will need to be seeded 2 or 3 d in advance to let them grow
Step 1B: approximately 1 h; however the mouse will need to be fasted from the night before
Step 1C: 1.5 h, depending on the amount of muscle to be minced
The goal of a mitochondrial preparation is to obtain a good amount of relatively pure, well coupled mitochondria. The quality of the obtained organelles can be checked by using oxygraphy to measure their oxygen consumption. For example, mitochondria isolated from mouse liver and energized with glutamate/malate respond to stimulation of ATPase by added ADP with a sixfold increase in the rate of oxygen consumption (Fig. 6b). This usually reflects mitochondria that are highly pure and intact. A closer look by conventional electron microscopy at the morphology and at the purity of the organelles isolated from other intracellular membranes revealed that most of the organelles displayed an intact inner and outer membrane and that the level of contamination by other membranes was kept to a minimum (Fig. 6a).
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.