This research was supported in part by NSF grant CHE-1229562 (VDGM), the Office of Undergraduate Research at Elon University, the Elon Chemistry Department, and the Elon Lumen Prize (TL and TAD), the Elon College Fellows Program (JAC), and the Elon College Honors Program (TAD).

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
<ol>
	<li>Cell culture
	<ol>
		<li>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 &#176;C in 5% CO2.</li>
		<li>Grow cells in T75 flasks and allow them to reach 90% confluence.&#160;</li>
		<li>Carry out cell splitting via trypsinization (0.25% Trypsin-EDTA) for 3-5 min at 37 &#176;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 &#176;C.</li>
		<li>Aspirate the media and resuspend the cell pellet in culture media.</li>
		<li>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.</li>
		<li>Serum starve cells 24 hr later by aspirating the media and replacing it with the same volume of serum-free media (DMEM, sterile).</li>
		<li>Prepare TNF-&#945;, IL1-&#946;, and IFN-&#947;, from lyophilized powder with ultra-pure water at working concentrations of 5 pg/&#181;l, 10 ng/&#181;l, and 10 ng/&#181;l, respectively and add them to the wells/flasks.</li>
		<li>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 &#8220;*3&#8221;), or sterile water as a control (referred to as &#8220;0&#8221;).</li>
		<li>Incubate treated cells for 24, 48 or 72 hr before assessment and harvesting.</li>
	</ol>
	</li>
	<li>Cell viability assessment
	<ol>
		<li>Make a 5 mg/ml solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in 1x PBS.</li>
		<li>For 12-well plates, add 100 &#181;l of MTT solution to each well and gently swirl to assure even distribution.</li>
		<li>Incubate plates for 15 min&#8211;1 hr at 37 &#176;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.</li>
		<li>Using a repeat pipettor, add 700 &#181;l 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 &#181;l of solvent and continue to swirl.</li>
		<li>For analysis, take 100 &#181;l 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.</li>
	</ol>
	</li>
	<li>Animal Model of ALS
	<ol>
		<li>
		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.
		</li>
		<li>Perform genotyping with standard primers against mutant SOD125.</li>
	</ol>
	</li>
</ol>
2. Isolation of Mitochondria
NOTE: It is important to work quickly and keep everything on ice throughout the procedure.
<ol>
	<li>Preparation of mitochondrial isolation buffer (MIB), spinal cord isolation buffer (SC MIB) and experimental buffer (EB)
	<ol>
		<li>Prepare the following stock solutions ahead of time for MIB and EB.</li>
		<li>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 &#176;C.</li>
		<li>Make 1 M Kphos by mixing 80.2 ml of K2HPO4 and 19.8 ml of KH2PO4. Store at room temperature.</li>
		<li>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.</li>
		<li>Make 1 M Glutamate by making 10 ml of 1 M solution of glutamic acid. Filter sterilize and store 4 &#176;C.</li>
		<li>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 &#176;C.</li>
		<li>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 &#176;C.</li>
		<li>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.</li>
		<li>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&#160;K phosphate, pH 7.4, and 10 mM EGTA/Tris. Filter sterilize and store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Equipment preparation prior to harvesting the cells.
	<ol>
		<li>Rinse a small glass vessel and homogenization pestle three times with sterile water and place on ice.</li>
		<li>Gather necessary items such as a standard drill, cell scrapers, 1.5 ml, 15 ml and 50 ml tubes and solutions.</li>
	</ol>
	</li>
	<li>Mitochondria Isolation
	<ol>
		<li>Cultured Cells
		<ol>
			<li>For each sample type, use two T175 flasks of cells.</li>
			<li>Ensure that cells are ~90% confluent and use in control experiments, or plate and treat as described above in step 1.2.</li>
			<li>On the bench top, aspirate media and wash the adherent cells twice with 15 ml of 1x PBS each time.</li>
			<li>Aspirate the buffer and scrape flasks to remove the adherent cells from the bottom of the flask.</li>
			<li>Add 15 ml of 1x PBS to each flask, swirl, and transfer to individual 15 ml conical tubes and place on ice.</li>
			<li>Once scraping is done, centrifuge the tubes for 5 min at 700 x g, 4 &#176;C using a table top centrifuge using a swinging bucket rotor.</li>
			<li>Aspirate supernatants and resuspend each pellet in 1 ml of MIB.</li>
			<li>Combine both suspensions and transfer to a small glass vessel for homogenization.</li>
			<li>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.</li>
			<li>Transfer the homogenized solution to a 50 ml conical tube.&#160;</li>
			<li>Draw the solution into a 3 ml syringe using an 18 gauge 1 &#189; inch needle and expel it back into the conical tube on ice with a 27 gauge &#189; inch needle.&#160;Take care to expel the solution against the inside wall of the tube as to utilize that force for cell membrane disruption.</li>
			<li>Repeat the syringe steps for a total of five times.&#160;</li>
			<li>Transfer the solution into a 15 ml conical tube and centrifuge for 5 min at 600 x g, 4 &#176;C in a table top centrifuge using a swinging bucket rotor.</li>
			<li>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.</li>
			<li>Centrifuge tubes in a fixed angle rotor at 10,000 x g, 4 &#176;C for 5 min.</li>
			<li>Aspirate supernatants and combine the pellets in 100 &#181;l 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.</li>
		</ol>
		</li>
		<li>Mitochondria isolation from mouse tissue
		<ol>
			<li>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%.</li>
			<li>Excise the liver and spinal cord from each animal and place separately in ice cold 1x PBS -/- to rinse away any blood.</li>
			<li>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.</li>
			<li>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.</li>
			<li>Transfer the homogenates to clean 15 ml tubes.</li>
			<li>Centrifuge tubes in a fixed angle rotor at 750 x g, 4 &#176;C for 10 min.</li>
			<li>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.</li>
			<li>Re-suspend the spinal cord pellets in 500 &#181;l of SC MIB.</li>
			<li>Re-homogenize each three times, only filling the vessel half way with SC MIB this time.</li>
			<li>Transfer the new homogenate to a fresh tube.</li>
			<li>Centrifuge the in a fixed angle rotor at 750 x g, 4 &#176;C for 10 min.</li>
			<li>Combine these new supernatants, which contain more mitochondria, with the first supernatant from each sample.</li>
			<li>Centrifuge tubes in a fixed angle rotor at 10,000 x g, 4 &#176;C for 5 min.</li>
			<li>Aspirate supernatants and resuspend the liver pellets in 500 &#181;l of MIB and the spinal cord pellets in 50 &#181;l 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.</li>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Cytochrome c assay using an R&#38;D Rat/Mouse Cytochrome c Quantikine ELISA Kit (adapted from the protocol provided by the manufacturer).
	<ol>
		<li>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 &#956;l of mitochondria per tube).</li>
		<li>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.</li>
		<li>Pellet mitochondria by centrifugation in a fixed angle rotor at 10,000 x g, 4 &#176;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 &#176;C for analysis later or used immediately.</li>
		<li>Determine release of cytochrome c by a comparing the concentration of cytochrome c in the pellet and supernatant27.
		<ol>
			<li>Prepare the samples by solubilizing the pellets in the original volume of the reaction with 0.5% Tx-100 in 1x PBS.</li>
			<li>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 &#956;l of cytochrome c conjugate (use straight out of bottle) plus 100 &#956;l of 0.5% Tx-100 in 1x PBS, and then all of the pellet or supernatant sample.</li>
			<li>Gently vortex the plate a low setting (level 2-4) for 20 sec to mix, cover and incubate for 1 hr, 37 &#176;C.</li>
			<li>Next, wash the plate four times with wash buffer diluted according to the manufacturer&#8217;s instructions. Remove any excess solution by tapping the wells on paper towel.</li>
			<li>Next, add 150 &#956;l of 1:1 A+B developing solution to each well and incubate the plate for 20-30 min in the dark at room temperature.</li>
			<li>Finally, add 50 &#956;l 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.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Assessment of Mitochondrial (Dys)function
<ol>
	<li>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 &#956;l of mitochondria are used per tube).</li>
	<li>Mitochondrial treatments
	<ol>
		<li>Add appropriate treatments (EB control, 1 &#956;M FCCP mixed with 50 nM Valinomycin, 10 &#956;M rotenone, 5 &#956;M oligomycin, 2 mM KCN, or 200 &#956;M ADP, final concentrations) to separate reactions at 1/10th 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.</li>
		<li>Dilute TMRE, reconstituted in sterile water at a working concentration of 100 &#956;M and stored at -20 &#176;C, to 2 &#956;M and add a volume equal to the mitochondrial reaction volume.</li>
		<li>Incubate the reactions on the bench top for 7 min.</li>
		<li>Pellet mitochondria by centrifugation in a fixed angle rotor at 10,000 x g, 4 &#176;C for 5 min.</li>
		<li>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&#8217;s instructions using the volume and plate that will be used for the assay. Using a standard 384-well black plate with 25 &#181;l of 1 &#181;M TMRE (final reaction concentration) the strongest signal was obtained using excitation/emission&#160;wavelengths&#160;485 nm/535 nm.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochemical+studies+of+mammalian+tissues;+isolation+of+intact+mitochondria+from+rat+liver;+some+biochemical+properties+of+mitochondria+and+submicroscopic+particulate+material.">Cytochemical studies of mammalian tissues; isolation of intact mitochondria from rat liver; some biochemical properties of mitochondria and submicroscopic particulate material.</a> The Journal Of Biological Chemistry. 172, 619-635 (1948).</li><li>Hogeboom, G. H., Claude, A., Hotch-Kiss, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+distribution+of+cytochrome+oxidase+and+succinoxidase+in+the+cytoplasm+of+the+mammalian+liver+cell.">The distribution of cytochrome oxidase and succinoxidase in the cytoplasm of the mammalian liver cell.</a> The Journal of Biological Chemistry. 165, 615-629 (1946).</li><li>Marques-Fernandez, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNFalpha+induces+survival+through+the+FLIP-L-dependent+activation+of+the+MAPK/ERK+pathway.">TNFalpha induces survival through the FLIP-L-dependent activation of the MAPK/ERK pathway.</a> Cell Death &amp; Disease. 4, e493 (2013).</li><li>Chao, C. C., Hu, S., Ehrlich, L., Peterson, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-1+and+tumor+necrosis+factor-alpha+synergistically+mediate+neurotoxicity:+involvement+of+nitric+oxide+and+of+N-methyl-D-aspartate+receptors.">Interleukin-1 and tumor necrosis factor-alpha synergistically mediate neurotoxicity: involvement of nitric oxide and of N-methyl-D-aspartate receptors.</a> Brain, Behavior, And Immunity. 9, 355-365 (1995).</li><li>Downen, M., Amaral, T. D., Hua, L. L., Zhao, M. L., Lee, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+death+in+cytokine-activated+primary+human+brain+cell+culture:+role+of+tumor+necrosis+factor-alpha.">Neuronal death in cytokine-activated primary human brain cell culture: role of tumor necrosis factor-alpha.</a> Glia. 28, 114-127 (1999).</li><li>Schildberger, A., Rossmanith, E., Weber, V., Falkenhagen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+endothelial+cell+activation+in+experimental+sepsis+with+a+two-step+cell+culture+model.">Monitoring of endothelial cell activation in experimental sepsis with a two-step cell culture model.</a> Innate Immunity. 16, 278-287 (2010).</li><li>Brown, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitrogen+disruption+of+synaptoneurosomes:+an+alternative+method+to+isolate+brain+mitochondria.">Nitrogen disruption of synaptoneurosomes: an alternative method to isolate brain mitochondria.</a> Journal Of Neuroscience Methods. 137, 299-303 (2004).</li><li>Wong, A., Cortopassi, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+measurement+of+mitochondrial+membrane+potential+in+a+neural+cell+line+using+a+fluorescence+plate+reader.">High-throughput measurement of mitochondrial membrane potential in a neural cell line using a fluorescence plate reader.</a> Biochemical and Biophysical Research Communications. 298, 750-754 (2002).</li><li>Blattner, J. R., He, L., Lemasters, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+assays+for+the+mitochondrial+permeability+transition+using+a+fluorescence+multiwell+plate+reader.">Screening assays for the mitochondrial permeability transition using a fluorescence multiwell plate reader.</a> Analytical Biochemistry. 295, 220-226 (2001).</li><li>Kataoka, M., Fukura, Y., Shinohara, Y., Baba, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+mitochondrial+membrane+potential+in+the+cells+by+microchip+flow+cytometry.">Analysis of mitochondrial membrane potential in the cells by microchip flow cytometry.</a> Electrophoresis. 26, 3025-3031 (2005).</li><li>Del Gaizo Moore, V., Payne, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transactivator+of+transcription+fusion+protein+transduction+causes+membrane+inversion.">Transactivator of transcription fusion protein transduction causes membrane inversion.</a> The Journal of Biological Chemistry. 279, 32541-32544 (2004).</li></ol>

The authors have nothing to disclose.

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 literature31,32. The ability to adjust the amounts and types of cytokine treatments as well as compariso...

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 (&#8710;&#968;)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 &#8710;&#968;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 apoptosome9,10. The apoptosome activates caspases that go on to cleave cellular proteins and DNA during the execution phase of apoptosis. As soon as MOMP occurs, &#8710;&#936; is collapsed and ATP production halted. Thus, as apoptosis is initiated mitochondrial function is compromised and changes in &#8710;&#936; can be correlated to mitochondrial and cell health12. While apoptosis is an endpoint in many disease models, mitochondrial function and changes to &#8710;&#936; 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 diseases13,14.
In the first part of the protocol, isolation of intact mitochondria that retain their &#916;&#936; is described. HEK-293T cells were exposed to different concentrations and combinations of recombinant TNF-&#945;, IL1-&#946; and IFN-&#947; 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-&#945; 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 &#916;&#936; 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 &#8710;&#936; 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 organelle16-18. 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 cultures13,14,19,20. 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 complexity22,23 and requires a substantial amount of mitochondria (~500 &#181;g/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 &#181;g/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.

<table><tbody><tr><td>L-glutamic acid</td><td>Sigma</td><td>G1251</td><td>Can use the potassium salt instead.</td></tr><tr><td>malic acid</td><td>Sigma</td><td>M8304</td><td>Can use the potassium salt instead.</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P0662</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P3786</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E3889</td><td></td></tr><tr><td>Trisma base</td><td>Sigma</td><td>T6066</td><td></td></tr><tr><td>MOPS</td><td>Sigma</td><td>M3183</td><td></td></tr><tr><td>CCCP</td><td>Sigma</td><td>C2920</td><td>Dilute down to 100&amp;nbsp;&amp;#956;M as a working stock in ethanol and store at -20&amp;nbsp;&amp;#176;C.</td></tr><tr><td>Valinomycin</td><td>Sigma</td><td>V0627</td><td>Make in DMSO and use as a 5&amp;nbsp;&amp;#956;M working stock. Store at -20&amp;nbsp;&amp;#176;C.</td></tr><tr><td>sucrose</td><td>Fisher</td><td>S5-500</td><td></td></tr><tr><td>KCN</td><td>Mallinckrodt</td><td>6379</td><td>Make a concentrated stock in ethanol and then dilute with water&amp;nbsp;</td></tr><tr><td>rotenone</td><td>Sigma</td><td>R8875</td><td>Highly toxic. Made in ethanol.</td></tr><tr><td>oligomycin</td><td>Sigma</td><td>O4876</td><td>Highly toxic. Made in ethanol.</td></tr><tr><td>ADP</td><td>Sigma</td><td>A2754</td><td></td></tr><tr><td>TMRE</td><td>Sigma</td><td>8717-25mg</td><td>Dilute 100&amp;nbsp;&amp;#956;M stock with EB immediatley before use.</td></tr><tr><td>DMEM</td><td>Gibco</td><td>11965-084</td><td>1x regular (high glucose).</td></tr><tr><td>Pen/Strep</td><td>Invitrogen</td><td>15140-155</td><td></td></tr><tr><td>L-glutamine</td><td>Fisher</td><td>SH3002101</td><td>Store aliquots at -20&amp;nbsp;&amp;#176;C</td></tr><tr><td>FBS</td><td>Lonza</td><td>14-501F</td><td>US origin, premium quality. Heat inactivate and store aliquots at -20&amp;nbsp;&amp;#176;C.</td></tr><tr><td>Trypsin-EDTA</td><td>Sigma</td><td>T4049</td><td></td></tr><tr><td>DMSO</td><td>SIgma</td><td>D2650</td><td></td></tr><tr><td>Protien Assay Dye (5x)</td><td>Bio-Rad</td><td>500-0006</td><td>Any protein assay can substitute.</td></tr><tr><td>BSA</td><td>Fisher</td><td>BP1600-100</td><td>Make 2 mg/ml stock in water for protein assay.</td></tr><tr><td>MTT powder</td><td>Sigma</td><td>M2128</td><td>Filter sterlize 5 mg/ml stock made in PBS. Store aliquots at -20&amp;nbsp;&amp;#176;C; store at 4&amp;nbsp;&amp;#176;C for up to 1 week.</td></tr><tr><td>Tergitol solution (NP-40)</td><td>Sigma</td><td>NP40S</td><td></td></tr><tr><td>Recombinant Human IL-1B</td><td>Gibco</td><td>PCH08014</td><td>Once opened store aliquots at -20&amp;nbsp;&amp;#176;C</td></tr><tr><td>Recombinant Human TNF-alpha</td><td>Gibco</td><td>PHC3015L</td><td></td></tr><tr><td>Recombinant Human IFN-gamma</td><td>Gibco</td><td>PHC4031</td><td></td></tr><tr><td>Dulbeccos PBS (-/-)</td><td>Sigma</td><td>D8537</td><td>Make sure it is without Mg&lt;sup&gt;2+&lt;/sup&gt; and Ca&lt;sup&gt;2+&lt;/sup&gt; ions.</td></tr><tr><td>Cytochrom &lt;em&gt;c&lt;/em&gt; ELISA kit</td><td>R&amp;amp;D systems</td><td>DTC0</td><td>Human for HEK-293T cells.&amp;nbsp;</td></tr></tbody></table>

isolation and functional analysis of mitochondria from cultured cells and mouse tissue

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.

Treatment of HEK-293T cells with 200 pg/ml TNF-&#945;, 40 ng/ml IL1-&#946;, and 75 ng/ml IFN-&#947; (*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-&#945;, 40 ng/ml IL1-&#946;, and 75 ng/ml IFN-&#947;) yield similar cell death results at 48 hr,...

Watch this Scientific Journal Video about Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue at JoVE.com

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

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.

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

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39.3K Views.  Elon University. 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. 

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

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are 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 cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Mitochondrial Fractionation in Small Tissue Samples for Supercomplex Analysis

Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial electron transport chain organization, giving rise to the proposal of the "plasticity model." This model postulates the coexistence of different proportions of SCs and complexes depending on the tissue or the cellular metabolic status. The dynamic nature of the assembly in SCs would allow cells to optimize the use of available fuels and the efficiency of electron transfer, minimizing reactive oxygen species generation and favoring the ability of cells to adapt to environmental changes. 
More recently, abnormalities in SC assembly have been reported in different diseases such as neurodegenerative disorders (Alzheimer's and Parkinson's disease), Barth Syndrome, Leigh syndrome, or cancer. The role of SC assembly alterations in disease progression still needs to be confirmed. Nevertheless, the availability of enough amounts of samples to determine the SC assembly status is often a challenge. This happens with biopsy or tissue samples that are small or have to be divided for multiple analyses, with cell cultures that have slow growth or come from microfluidic devices, with some primary cultures or rare cells, or when the effect of particular costly treatments has to be analyzed (with nanoparticles, very expensive compounds, etc.). In these cases, an efficient and easy-to-apply method is required. This paper presents a method adapted to obtain enriched mitochondrial fractions from small amounts of cells or tissues to analyze the structure and function of mitochondrial SCs by native electrophoresis followed by in-gel activity assays or western blot.

Author Spotlight: Unveiling Oxidative Phosphorylation System Dynamics and Mitochondrial Roles in Health and Disease

This protocol describes a technique for the analysis of respiratory supercomplexes when only small amounts of samples are available.

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial ...

Biochemistry

Mitochondrial Isolation from Rodents for High-Quality Bioenergetic Studies

Robust Mitochondrial Isolation from Rodent Cardiac Tissue

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry to study metabolic impairments and disease. Consistent mitochondrial quality is necessary to properly investigate mitochondrial physiology and bioenergetics; however, many different published isolation methods are available for researchers. Although different experimental strategies require different isolation methods, the basic principles and procedures are similar. This protocol details a method capable of extracting well-coupled mitochondria from a variety of tissue sources, including small animals and cells. The steps outlined include organ dissection, mitochondrial purification, protein quantification, and various quality control checks. The primary quality control metric used to identify high-quality mitochondria is the respiratory control ratio (RCR). The RCR is the ratio of the respiratory rate during oxidative phosphorylation to the rate in the absence of ADP. Alternative metrics are discussed. While high RCR values relative to their tissue source are obtained using this protocol, several steps can be optimized to suit the individual needs of researchers. This procedure is robust and has consistently resulted in isolated mitochondria with above-average RCR values across animal models and tissue sources.

Author Spotlight: Uncovering the Role of Mitochondrial Calcium Phosphate in Heart Failure and Bioenergetics

Bioenergetic and metabolomic studies on mitochondria have revealed their multifaceted role in many diseases, but the isolation methods for these organelles vary. The method detailed here is capable of purifying high-quality mitochondria from multiple tissue sources. Quality is determined by respiratory control ratios and other metrics assessed with high-resolution respirometry.

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential ...

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in&#160;any given organ or tissue.
Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...

Improved Protein Quantification Using Two-Step Mitochondrial Isolation

Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification

Most physiological and disease processes, from central metabolism to immune response to neurodegeneration, involve mitochondria. The mitochondrial proteome is composed of more than 1,000 proteins, and the abundance of each can vary dynamically in response to external stimuli or during disease progression. Here, we describe a protocol for isolating high-quality mitochondria from primary cells and tissues. The two-step procedure comprises (1) mechanical homogenization and differential centrifugation to isolate crude mitochondria, and (2) tag-free immune capture of mitochondria to isolate pure organelles and eliminate contaminants. Mitochondrial proteins from each purification stage are analyzed by quantitative mass spectrometry, and enrichment yields are calculated, allowing the discovery of novel mitochondrial proteins by subtractive proteomics. Our protocol provides a sensitive and comprehensive approach to studying mitochondrial content in cell lines, primary cells, and tissues.

Author Spotlight: Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification  

We present a two-step protocol for high-quality mitochondria isolation that is compatible with protein discovery and quantification at a proteome scale. Our protocol does not require genetic engineering and is thus suitable for studying mitochondria from any primary cells and tissues.