Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Podsumowanie

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Streszczenie

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Wprowadzenie

Living cells bank metabolic energy in the form of fats and carbohydrates and use this energy for biosynthesis, membrane transport and movement. Some energy is obtained in the cytosol through the conversion of dietary sugars directly into ATP during glycolysis. However, the main source of ATP production in a cell is harnessed within the mitochondria via the mitochondrial respiratory chain¹. The architecture of mitochondria provides the necessary spatial orientation for effective and efficient ATP production. Mitochondria possess a double membrane separated by an intermembrane space and together with the matrix, the innermost mitochondrial compartment, house the components and coordinate the chemical reactions involved in ATP generation. The inner membrane contains a series of membrane-bound protein complexes that comprise the respiratory chain as well as the ATP synthase, the protein complex that brings ADP and Pi together for formation of ATP. The inner membrane is folded into cristae and electrons are passed along the respiratory chain complexes via cytochrome c, a soluble electron carrier which moves between complexes within the intermembrane space. As electrons move, oxidation of reducing equivalents occurs and hydrogen ions are pumped from the matrix to the intermembrane space. As a consequence of the high ion concentration within the intermembrane space, an electrochemical gradient builds resulting in a membrane potential across the inner mitochondrial membrane (∆ψ)². Oxygen is the final electron acceptor of the electron transport chain, and hydrogen ions flow through the ATP synthases from the intermembrane space back to the matrix, and in doing so directly cause ATP formation. This process as described in its entirety is known as oxidative phosphorylation. The folds of the cristae increase the surface area of the inner membrane, allowing for maximal electron transport and ATP production within each mitochondrion. The proteins, enzymes and other molecules involved in oxidative phosphorylation are derived from both nuclear and mitochondrial genes. Mitochondria contain their own circular DNA, encoding for 13 proteins as well as tRNAs and mRNAs necessary for ATP production³. However, many more proteins are required and thus are nuclear encoded. Most of these nuclear-encoded proteins are targeted to the mitochondrial matrix by the use of presequences at the N-terminus of the precursor protein, and their import is driven in part by ∆ψ^4,5.

Beyond contributing to the bioenergetics of a cell, mitochondria also influence major metabolic processes such as TCA and beta-oxidation, cellular signaling through regulating calcium, as well as a key role in apoptosis⁶. Specifically, during times of cellular stress, BCL-2 family proteins that reside on or interact at the mitochondrial outer membrane can cause mitochondrial outer membrane permeability (MOMP)^7,8. During MOMP, cytochrome c and other proteins are released into the cytosol, and together with several cytosolic proteins form a complex called the apoptosome^9,10. The apoptosome activates caspases that go on to cleave cellular proteins and DNA during the execution phase of apoptosis. As soon as MOMP occurs, ∆Ψ is collapsed and ATP production halted. Thus, as apoptosis is initiated mitochondrial function is compromised and changes in ∆Ψ can be correlated to mitochondrial and cell health¹². While apoptosis is an endpoint in many disease models, mitochondrial function and changes to ∆Ψ can also yield valuable information about disease origination and/or progression. For instance, mitochondrial structural and functional changes have been documented during the course of neurodegenerative diseases^13,14.

In the first part of the protocol, isolation of intact mitochondria that retain their ΔΨ is described. HEK-293T cells were exposed to different concentrations and combinations of recombinant TNF-α, IL1-β and IFN-γ to induce apoptosis. These cytokines were chosen because they are frequently reported to be high in primary human septic samples¹⁵ and the extrinsic pathway of apoptosis can be triggered by interaction of TNF-α binding to its receptor⁶ . Since there are subtle variations necessary to isolate functional mitochondria from primary tissues as compared to cultured cells, and because much research utilizes animals, the protocol also describes how to isolate mitochondria from liver and spinal cords of anterior lateral sclerosis (ALS) mouse model.

The second part of the protocol was developed to monitor perturbations to the mitochondrial membrane potential using a potential-sensitive fluorescent dye with a fluorescent plate reader. Differences between cellular status (i.e., healthy vs. unhealthy) are differentiated by quantitating the strength of ΔΨ of isolated mitochondria in conjunction with uncoupling agents, respiratory chain inhibitors, complex inhibitors and ionophores, all of which cause dissipation of the mitochondrial membrane potential. The healthier the mitochondria, the larger the change in ∆Ψ upon treatment with mitochondrial inhibitors, therefore the response of mitochondria can be used as an indicator of mitochondrial (dys)function.

Use of isolated mitochondria rather than in situ assessment of function offers definitive evidence that a pathology or treatment directly modulates changes to the organelle^16-18. While there are methods in the literature to isolate mitochondria from cultured cells, they are vague¹⁷ and/or utilize specialized equipment¹⁶. This protocol describes in detail the isolation method and is readily adaptable to other cell lines, including primary tissue and cultures^13,14,19,20. Many isolated mitochondria studies utilize the same mitochondrial uncouplers and inhibitors used in this protocol but with a Clark electrode (²¹ is a representative example of many papers in the literature), which again is a very specific and specialized piece of equipment. Furthermore, this traditional method has limitations such as low throughput and high complexity^22,23 and requires a substantial amount of mitochondria (~500 µg/reaction). In this protocol, the fluorescent membrane-sensing potential probe TMRE is used in conjunction with a fluorescent plate reader, which is a standard machine in many laboratories. TMRE is widely regarded since it quickly enters cells and isolated mitochondria and can be used at low concentrations²⁴. Multiple reactions can quickly be set up in tandem and batch analyzed using this protocol. Furthermore, the reactions require a much small amount of isolated mitochondria (~10 µg/reaction). By requiring less material, smaller tissue or cell culture samples can be utilized as a starting point for mitochondria isolation, more replicates or reactions can be set up, and potentially enough material for other isolated mitochondria experiments such as ATP production, oxygen consumption, or import assays are possible.

Protokół

All animal experiments conformed to National Institutes of Health guidelines and were approved by the Wake Forest University Animal Care and Use Committee. Breeding pairs for SOD1G93A [B6SJL-TgN (SOD1-G93A) 1Gur] mouse model were obtained from The Jackson Laboratory (Bar Harbor, ME). Nontransgenic wild-type (WT) females and SOD1G93A males [B6SJL-TgN (SOD1-G93A) 1Gur] were bred to generate SOD1G93A mice and nontransgenic WT littermates that were used in the experiments.

1. Models for Investigation

1. Cell culture
   1. Maintain human embryonic kidney cells (HEK-T293) cells in DMEM supplemented with 10% heat inactivated fetal bovine serum, 1% penicillin-streptomycin, and 1% L-glutamine, at 37 °C in 5% CO₂.
   2. Grow cells in T75 flasks and allow them to reach 90% confluence. 
   3. Carry out cell splitting via trypsinization (0.25% Trypsin-EDTA) for 3-5 min at 37 °C in 5% CO₂, inactivation of trypsin with equal volume of cell media, and centrifugation of cells for 5 min at 700 x g in 4 °C.
   4. Aspirate the media and resuspend the cell pellet in culture media.
   5. Seed cells in the appropriate flask or plate 7 x 10⁴ cells into a T75 flask 48 hr prior to cytokine treatment. This should give ~70% density at time of treatment.
   6. Serum starve cells 24 hr later by aspirating the media and replacing it with the same volume of serum-free media (DMEM, sterile).
   7. Prepare TNF-α, IL1-β, and IFN-γ, from lyophilized powder with ultra-pure water at working concentrations of 5 pg/µl, 10 ng/µl, and 10 ng/µl, respectively and add them to the wells/flasks.
   8. Treat serum starved cells by the addition of solubilized cytokines directly to the cell culture media of appropriate flasks. Treatments consisted of individual cytokines, a cocktail of all 3 (referred to as “*3”), or sterile water as a control (referred to as “0”).
   9. Incubate treated cells for 24, 48 or 72 hr before assessment and harvesting.
2. Cell viability assessment
   1. Make a 5 mg/ml solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in 1x PBS.
   2. For 12-well plates, add 100 µl of MTT solution to each well and gently swirl to assure even distribution.
   3. Incubate plates for 15 min–1 hr at 37 °C in 5% CO₂, and check under a microscope for the presence of purple coloration. If no purple is present, swirl again and allow cells to incubate in 5 min intervals until purple is observed. The amount of time necessary should be determined by each investigator.
   4. Using a repeat pipettor, add 700 µl of MTT solvent (4 mM HCl and 0.1% NP40 in isopropanol) to each well and rock the plate(s) at room temperature for 5-10 min, or until all purple color had left the cells. If purple color does not come out of cells, add an additional 200 µl of solvent and continue to swirl.
   5. For analysis, take 100 µl from each well and place into a 96-well plate and read on a plate reader using 570 nm and a reference wavelength of 630 nm, however 595 nm is also acceptable as long as readings are taken at consistent settings.
3. Animal Model of ALS
   1. All animal experiments conformed to National Institutes of Health guidelines and were approved by the Wake Forest University Animal Care and Use Committee. Breeding pairs for SOD1G93A [B6SJL-TgN (SOD1-G93A) 1Gur] mouse model were obtained from The Jackson Laboratory (Bar Harbor, ME). Nontransgenic wild-type (WT) females and SOD1G93A males [B6SJL-TgN (SOD1-G93A) 1Gur] were bred to generate SOD1G93A mice and nontransgenic WT littermates that were used in the experiments.
   2. Perform genotyping with standard primers against mutant SOD1²⁵.

2. Isolation of Mitochondria

NOTE: It is important to work quickly and keep everything on ice throughout the procedure.

1. Preparation of mitochondrial isolation buffer (MIB), spinal cord isolation buffer (SC MIB) and experimental buffer (EB)
   1. Prepare the following stock solutions ahead of time for MIB and EB.
   2. Make 1 M Tris/MOPS by dissolving 12.1 g of Tris base in 70 ml of H₂O. Add dry MOPS to get pH to 7.4. Adjust volume to 100 ml final. Filter sterilize and store at 4 °C.
   3. Make 1 M Kphos by mixing 80.2 ml of K₂HPO₄ and 19.8 ml of KH₂PO₄. Store at room temperature.
   4. Make 0.2 M EGTA/Tris by adding 3.8 g of EGTA to 10 ml of H₂O. Add 1 M Tris/MOPS until dissolved, ~30-40 ml. Adjust to 50 ml, sterile filter and store at room temperature. Note that the pH will be ~6.7.
   5. Make 1 M Glutamate by making 10 ml of 1 M solution of glutamic acid. Filter sterilize and store 4 °C.
   6. Make 1 M Malate by making 10 ml of 1 M solution of malic acid. Add Tris/MOPS to 50 mM. Filter sterilize and store at 4 °C.
   7. Make MIB at a concentration of 200 mM sucrose, 10 mM Tris/MOPS, pH 7.4, and 1 mM EGTA/Tris. Filter sterilize and store at 4 °C.
   8. Make SC MIB at a concentration of 250 mM sucrose, 20 mM HEPES-KOH pH 7.5, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and protease inhibitor cocktail.
   9. Make EB at a concentration of 125 mM KCl, 10 mM Tris/MOPS, pH 7.4, 5 mM glutamate, 2.5 mM malate, 1 mM K phosphate, pH 7.4, and 10 mM EGTA/Tris. Filter sterilize and store at 4 °C.
2. Equipment preparation prior to harvesting the cells.
   1. Rinse a small glass vessel and homogenization pestle three times with sterile water and place on ice.
   2. Gather necessary items such as a standard drill, cell scrapers, 1.5 ml, 15 ml and 50 ml tubes and solutions.
3. Mitochondria Isolation
   1. Cultured Cells
      1. For each sample type, use two T175 flasks of cells.
      2. Ensure that cells are ~90% confluent and use in control experiments, or plate and treat as described above in step 1.2.
      3. On the bench top, aspirate media and wash the adherent cells twice with 15 ml of 1x PBS each time.
      4. Aspirate the buffer and scrape flasks to remove the adherent cells from the bottom of the flask.
      5. Add 15 ml of 1x PBS to each flask, swirl, and transfer to individual 15 ml conical tubes and place on ice.
      6. Once scraping is done, centrifuge the tubes for 5 min at 700 x g, 4 °C using a table top centrifuge using a swinging bucket rotor.
      7. Aspirate supernatants and resuspend each pellet in 1 ml of MIB.
      8. Combine both suspensions and transfer to a small glass vessel for homogenization.
      9. Add MIB to the vessel until buffer reaches the first line. Homogenize cells with the pestle attached to a drill at medium speed for three passes. Ensure that the vessel is on ice during this step, not to remove the pestle above the liquid and to use a steady speed with continuous passes.
      10. Transfer the homogenized solution to a 50 ml conical tube. 
      11. Draw the solution into a 3 ml syringe using an 18 gauge 1 ½ inch needle and expel it back into the conical tube on ice with a 27 gauge ½ inch needle. Take care to expel the solution against the inside wall of the tube as to utilize that force for cell membrane disruption.
      12. Repeat the syringe steps for a total of five times. 
      13. Transfer the solution into a 15 ml conical tube and centrifuge for 5 min at 600 x g, 4 °C in a table top centrifuge using a swinging bucket rotor.
      14. Carefully remove the supernatant and distribute among three 1.5 ml tubes.
          NOTE: Mitochondria are in the supernatant after this first, low-speed spin, while cell membranes and unbroken cells are pelleted.
      15. Centrifuge tubes in a fixed angle rotor at 10,000 x g, 4 °C for 5 min.
      16. Aspirate supernatants and combine the pellets in 100 µl MIB and immediately place on ice.
          NOTE: Mitochondria are in the pellet after this high-speed spin. Mitochondria will typically maintain their membrane potential for ~2-3 ion ice after isolation and are most stable as a concentrated stock solution in MIB.
   2. Mitochondria isolation from mouse tissue
      1. Anesthetize the mouse according to IACUC protocol. Set a vaporizer at 5.0% to induce anesthesia. Confirmed that the animal is anesthetized by a lack of reflex for foot pinch or blink reflex when the eye is approached or tapped with a cotton swab. Keep the mouse under anesthesia for the entire surgical procedure by keeping the vaporizer between 1.5% and 2.0%.
      2. Excise the liver and spinal cord from each animal and place separately in ice cold 1x PBS -/- to rinse away any blood.
      3. For the liver, transfer the tissue to a weigh dish on ice and chop into fine pieces using a fresh razor blade for 1 min.
      4. Add tissue and appropriate buffer (MIB for liver or SC MIB for the spinal cord) to the vessel until buffer reaches the first line. Homogenize tissue with the pestle by hand for five passes. Ensure that the vessel is on ice during this step, not to remove the pestle above the liquid and to use a steady speed with continuous passes.
      5. Transfer the homogenates to clean 15 ml tubes.
      6. Centrifuge tubes in a fixed angle rotor at 750 x g, 4 °C for 10 min.
      7. Save the supernatants into clean tubes and place them on ice.
         NOTE: Mitochondria are in the supernatant after this first, low-speed spin, while cell membranes and unbroken cells are pelleted.
      8. Re-suspend the spinal cord pellets in 500 µl of SC MIB.
      9. Re-homogenize each three times, only filling the vessel half way with SC MIB this time.
      10. Transfer the new homogenate to a fresh tube.
      11. Centrifuge the in a fixed angle rotor at 750 x g, 4 °C for 10 min.
      12. Combine these new supernatants, which contain more mitochondria, with the first supernatant from each sample.
      13. Centrifuge tubes in a fixed angle rotor at 10,000 x g, 4 °C for 5 min.
      14. Aspirate supernatants and resuspend the liver pellets in 500 µl of MIB and the spinal cord pellets in 50 µl SC MIB and immediately place on ice.
          NOTE: Mitochondria are in the pellet after this high-speed spin. Mitochondria will typically maintain their membrane potential for ~2-3 ion ice after isolation and are most stable as a concentrated stock solution in MIB.
      15. Perform a protein concentration assay to estimate the concentration of mitochondria in solution. Follow instructions for using a commercial Protein Assay Kit or similar method²⁶. Typical concentrations of mitochondria are: 2 T175 flasks yields ~2 mg/ml, 1 mouse liver ~3-5 mg/ml, and 1 mouse spinal cord ~1-3 mg/ml.
4. Cytochrome c assay using an R&D Rat/Mouse Cytochrome c Quantikine ELISA Kit (adapted from the protocol provided by the manufacturer).
   1. Immediately before setting up the reactions, dilute mitochondria to 0.5 mg/ml working concentration with EB and distribute diluted mitochondria in 1.5 ml tubes for reactions (typically 30-50 μl of mitochondria per tube).
   2. Add either EB or DMSO, at 1% of the total volume, to the diluted mitochondria and incubate the reactions on the bench top for 7-10 min. These reactions are stable for up to 30 min at room temperature if a longer time frame is needed.
   3. Pellet mitochondria by centrifugation in a fixed angle rotor at 10,000 x g, 4 °C for 5 min and carefully separate the supernatant and place each in a separate 1.5 ml tubes.
      NOTE: Tubes can either be frozen at -20 °C for analysis later or used immediately.
   4. Determine release of cytochrome c by a comparing the concentration of cytochrome c in the pellet and supernatant²⁷.
      1. Prepare the samples by solubilizing the pellets in the original volume of the reaction with 0.5% Tx-100 in 1x PBS.
      2. For each sample, prepare 2 wells on the ELISA plate, one for the now solubilized pellet and one for the supernatant from the reaction by addition of 100 μl of cytochrome c conjugate (use straight out of bottle) plus 100 μl of 0.5% Tx-100 in 1x PBS, and then all of the pellet or supernatant sample.
      3. Gently vortex the plate a low setting (level 2-4) for 20 sec to mix, cover and incubate for 1 hr, 37 °C.
      4. Next, wash the plate four times with wash buffer diluted according to the manufacturer’s instructions. Remove any excess solution by tapping the wells on paper towel.
      5. Next, add 150 μl of 1:1 A+B developing solution to each well and incubate the plate for 20-30 min in the dark at room temperature.
      6. Finally, add 50 μl of stop solution to each well and take absorbance readings at 540 nm using a microplate reader. Calculate the amount of cytochrome c release by comparing the amount of cytochrome c in the pellet vs. supernatant for each reaction.

3. Assessment of Mitochondrial (Dys)function

1. Immediately before reactions are set up, dilute mitochondria to 0.5 mg/ml working concentration with EB and placed in 1.5 ml tubes for reactions (typically 30-50 μl of mitochondria are used per tube).
2. Mitochondrial treatments
   1. Add appropriate treatments (EB control, 1 μM FCCP mixed with 50 nM Valinomycin, 10 μM rotenone, 5 μM oligomycin, 2 mM KCN, or 200 μM ADP, final concentrations) to separate reactions at 1/10^th the total volume of diluted mitochondria. Incubate the reactions on the bench top for 7 min.
      NOTE: Care should be taken when handling these substances as accidental ingestion or absorption through the skin could be harmful.
   2. Dilute TMRE, reconstituted in sterile water at a working concentration of 100 μM and stored at -20 °C, to 2 μM and add a volume equal to the mitochondrial reaction volume.
   3. Incubate the reactions on the bench top for 7 min.
   4. Pellet mitochondria by centrifugation in a fixed angle rotor at 10,000 x g, 4 °C for 5 min.
   5. Load half of the supernatant volume of each sample onto a 396-well plate and read fluorescence.
      NOTE: Excitation and emission wavelengths of TMRE should be optimized according to manufacturer’s instructions using the volume and plate that will be used for the assay. Using a standard 384-well black plate with 25 µl of 1 µM TMRE (final reaction concentration) the strongest signal was obtained using excitation/emission wavelengths 485 nm/535 nm.
   6. Calculate the fold-difference in fluorescence between control (EB) and each treatment by dividing the relative fluorescence value (RFU) obtained from the plate reader for the experimental sample by the RFU from the control sample.

Wyniki

Treatment of HEK-293T cells with 200 pg/ml TNF-α, 40 ng/ml IL1-β, and 75 ng/ml IFN-γ (*3) for 24-48 hr leads to progressive amounts of cell death (Figure 1A). Cell viability was assessed using MTT assays and consistently demonstrates that there is ~10% decrease in cell viability with 24 hr treatment and ~20% decrease with 48 hr treatment. Cells treated with similar concentrations (100 pg/ml TNF-α, 40 ng/ml IL1-β, and 75 ng/ml IFN-γ) yield similar cell death results at 48 hr,...

Dyskusje

Treatment of HEK-293T cells with recombinant cytokines causes moderate amounts of cell death over 48 hr (Figure 1). The amount of cell death induced by TNF-alpha treatment is similar to previously reported studies³⁰ and cell viability decreases after co-administration of multiple cytokines that are greater than summative amounts with any cytokine alone is also consistent with the literature^31,32. The ability to adjust the amounts and types of cytokine treatments as well as compariso...

Materiały

Name	Company	Catalog Number	Comments
L-glutamic acid	Sigma	G1251	Can use the potassium salt instead.
malic acid	Sigma	M8304	Can use the potassium salt instead.
KH2PO4	Sigma	P0662
K2HPO4	Sigma	P3786
EGTA	Sigma	E3889
Trisma base	Sigma	T6066
MOPS	Sigma	M3183
CCCP	Sigma	C2920	Dilute down to 100 μM as a working stock in ethanol and store at -20 °C.
Valinomycin	Sigma	V0627	Make in DMSO and use as a 5 μM working stock. Store at -20 °C.
sucrose	Fisher	S5-500
KCN	Mallinckrodt	6379	Make a concentrated stock in ethanol and then dilute with water
rotenone	Sigma	R8875	Highly toxic. Made in ethanol.
oligomycin	Sigma	O4876	Highly toxic. Made in ethanol.
ADP	Sigma	A2754
TMRE	Sigma	8717-25mg	Dilute 100 μM stock with EB immediatley before use.
DMEM	Gibco	11965-084	1x regular (high glucose).
Pen/Strep	Invitrogen	15140-155
L-glutamine	Fisher	SH3002101	Store aliquots at -20 °C
FBS	Lonza	14-501F	US origin, premium quality. Heat inactivate and store aliquots at -20 °C.
Trypsin-EDTA	Sigma	T4049
DMSO	SIgma	D2650
Protien Assay Dye (5x)	Bio-Rad	500-0006	Any protein assay can substitute.
BSA	Fisher	BP1600-100	Make 2 mg/ml stock in water for protein assay.
MTT powder	Sigma	M2128	Filter sterlize 5 mg/ml stock made in PBS. Store aliquots at -20 °C; store at 4 °C for up to 1 week.
Tergitol solution (NP-40)	Sigma	NP40S
Recombinant Human IL-1B	Gibco	PCH08014	Once opened store aliquots at -20 °C
Recombinant Human TNF-alpha	Gibco	PHC3015L
Recombinant Human IFN-gamma	Gibco	PHC4031
Dulbeccos PBS (-/-)	Sigma	D8537	Make sure it is without Mg2+ and Ca2+ ions.
Cytochrom c ELISA kit	R&D systems	DTC0	Human for HEK-293T cells.