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 border="0" cellpadding="0" cellspacing="0" width="686">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Name of Material/ Equipment</td>
			<td style="width:137px;">Company</td>
			<td style="width:163px;">Catalog Number</td>
			<td style="width:179px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-glutamic acid</td>
			<td>Sigma</td>
			<td>G1251</td>
			<td>Can use the potassium salt instead.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">malic acid</td>
			<td>Sigma</td>
			<td>M8304</td>
			<td>Can use the potassium salt instead.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KH2PO4</td>
			<td>Sigma</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">K2HPO4</td>
			<td>Sigma</td>
			<td>P3786</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGTA</td>
			<td>Sigma</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trisma base</td>
			<td>Sigma</td>
			<td>T6066</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MOPS</td>
			<td>Sigma</td>
			<td>M3183</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CCCP</td>
			<td>Sigma</td>
			<td>C2920</td>
			<td>Dilute down to 100uM as a working stock in ethanol and store at -20C.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Valinomycin</td>
			<td>Sigma</td>
			<td>V0627</td>
			<td>Make in DMSO and use as a 5uM working stock. Store at -20C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sucrose</td>
			<td>Fisher</td>
			<td>S5-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCN</td>
			<td>Mallinckrodt</td>
			<td align="right">6379</td>
			<td>Make a concentrated stock in ethanol and then dilute with water&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">rotenone</td>
			<td>Sigma</td>
			<td>R8875</td>
			<td>Highly toxic. Made in ethanol.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">oligomycin</td>
			<td>Sigma</td>
			<td>O4876</td>
			<td>Highly toxic. Made in ethanol.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ADP</td>
			<td>Sigma</td>
			<td>A2754</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TMRE</td>
			<td>Sigma</td>
			<td>8717-25mg</td>
			<td>Dilute 100uM stock with EB immediatley before use.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>Gibco</td>
			<td>11965-084</td>
			<td>1x regular (high glucose).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pen/Strep</td>
			<td>Invitrogen</td>
			<td>15140-155</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-glutamine</td>
			<td>Fisher</td>
			<td>SH3002101</td>
			<td>Store aliquots at -20C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS</td>
			<td>Lonza</td>
			<td>14-501F</td>
			<td>US origin, premium quality. Heat inactivate and store aliquots at -20</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA</td>
			<td>Sigma</td>
			<td>T4049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMSO</td>
			<td>SIgma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">Protien Assay Dye (5x)</td>
			<td style="width:137px;">Bio-Rad</td>
			<td style="width:163px;">500-0006</td>
			<td>Any protein assay can substitute.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">BSA</td>
			<td style="width:137px;">Fisher</td>
			<td style="width:163px;">BP1600-100</td>
			<td>Make 2mg/mL stock in water for protein assay.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MTT powder</td>
			<td>Sigma</td>
			<td>M2128</td>
			<td>Filter sterlize 5mg/mL stock made in PBS. Store aliquots at -20C; store at 4C for up to 1 week.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tergitol solution (NP-40)</td>
			<td>Sigma</td>
			<td>NP40S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Recombinant Human IL-1B</td>
			<td>Gibco</td>
			<td>PCH08014</td>
			<td>Once opened store aliquots at -20C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Recombinant Human TNF-alpha</td>
			<td>Gibco</td>
			<td>PHC3015L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant Human IFN-gamma</td>
			<td style="width:137px;">Gibco</td>
			<td>PHC4031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">Dulbeccos PBS (-/-)</td>
			<td>Sigma</td>
			<td style="width:163px;">D8537</td>
			<td>Make sure it is without Mg2+ and Ca2+ ions.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cytochrom c ELISA kit</td>
			<td>R&#38;D systems</td>
			<td>DTC0</td>
			<td>Human for HEK-293T cells.&#160;</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 ...

isolation-functional-analysis-mitochondria-from-cultured-cells-mouse

Research

JoVE Journal

Biology

39.2K 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 ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

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 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 ...

Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma

Cerebral blood flow is conveyed by vascular resistance arteries and downstream parenchymal arterioles. Steady-state vascular resistance to blood flow increases with decreasing diameter from arteries to arterioles that ultimately feed into capillaries. Due to their smaller size and location in the parenchyma, arterioles have been relatively understudied and with less reproducibility in findings than surface pial arteries. Regardless, arteriolar endothelial cell structure and function&#8212;integral to the physiology and etiology of chronic degenerative diseases&#8212;requires extensive investigation. In particular, emerging evidence demonstrates that compromised endothelial function precedes and exacerbates cognitive impairment and dementia.
In the parenchymal microcirculation, endothelial K+ channel function is the most robust stimulus to finely control the spread of vasodilation to promote increases in blood flow to areas of neuronal activity. This paper illustrates a refined method for freshly isolating intact and electrically coupled endothelial &#34;tubes&#34; (diameter, ~25 &#181;m) from mouse brain parenchymal arterioles. Arteriolar endothelial tubes are secured during physiological conditions (37 &#176;C, pH 7.4) to resolve experimental variables that encompass K+ channel function and their regulation, including intracellular Ca2+ dynamics, changes in membrane potential, and membrane lipid regulation. A distinct technical advantage versus arterial endothelium is the enhanced morphological resolution of cell and organelle (e.g., mitochondria) dimensions, which expands the usefulness of this technique. Healthy cerebral perfusion throughout life entails robust endothelial function in parenchymal arterioles, directly linking blood flow to the fueling of neuronal and glial activity throughout precise anatomical regions of the brain. Thus, it is expected that this method will significantly advance the general knowledge of vascular physiology and neuroscience concerning the healthy and diseased brain.

Intensive preparation of intact mouse cerebral endothelial &#34;tubes&#34; from cerebral parenchymal arterioles is illustrated for studying cerebral blood flow regulation. Further, we demonstrate the experimental strengths of this endothelial study model for fluorescence imaging and electrophysiology measurement of key cellular signaling pathways, including changes in intracellular [Ca2+] and membrane potential.

Cerebral blood flow is conveyed by vascular resistance arteries and downstream parenchymal arterioles. Steady-state vascular resistance to blood flow ...

Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria

Glomerular endothelial cell (GEC) dysfunction can initiate and contribute to glomerular filtration barrier breakdown. Increased mitochondrial oxidative stress has been suggested as a mechanism resulting in GEC dysfunction in the pathogenesis of some glomerular diseases. Historically the isolation of GECs from in vivo models has been notoriously challenging due to difficulties in isolating pure cultures from glomeruli. GECs have complex growth requirements in vitro and a very limited lifespan. Here, we describe the procedure for isolating and culturing conditionally immortalized GECs with fluorescent mitochondria, enabling the tracking of mitochondrial fission and fusion events. GECs were isolated from the kidneys of a double transgenic mouse expressing the thermolabile SV40 TAg (from the Immortomouse), conditionally promoting proliferation and suppressing cell differentiation, and a photo-convertible fluorescent protein (Dendra2) in all mitochondria (from the photo-activatable mitochondria [PhAMexcised] mouse). The stable cell line generated allows for cell differentiation after inactivation of the immortalizing SV40 TAg gene and photo-activation of a subset of mitochondria causing a switch in fluorescence from green to red. The use of mitoDendra2-GECs allows for live imaging of fluorescent mitochondria&#39;s distribution, fusion, and fission events without staining the cells.

The article describes the method for isolating conditionally immortalized glomerular endothelial cells from the kidneys of transgenic mice expressing the thermolabile simian virus 40 and photo-activatable mitochondria, PhAMexcised. We describe the procedure for glomeruli isolation from whole kidneys using beads, digestion steps, seeding, and culturing of GECs-CD31 positive.

Glomerular endothelial cell (GEC) dysfunction can initiate and contribute to glomerular filtration barrier breakdown. Increased mitochondrial oxidative ...

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important players in the maintenance of organismal homeostasis and the progression of a wide spectrum of diseases. While the current research focuses on the characterization of endogenous exosomes with the endosomal origin, microvesicles blebbing from the plasma membrane have gained increasing attention in health and sickness, which are featured by an abundance of surface molecules recapitulating the membrane signature of parent cells. Here, a reproducible procedure is presented based on differential centrifugation for extracting and characterizing EVs from the plasma and solid tissues, such as the bone. The protocol further describes subsequent profiling of surface antigens and protein cargos of EVs, which are thus traceable for their derivations and identified with components related to potential function. This method will be useful for correlative, functional, and mechanistic analysis of EVs in biological, physiological, and pathological studies.

The present protocol describes a method to extract extracellular vesicles from the peripheral blood and solid tissues with subsequent profiling of surface antigens and protein cargos.

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important ...