This work was supported by grant number &#34;PGC2018-095795-B-I00&#34; from Ministerio de Ciencia e Innovaci&#243;n (https://ciencia.sede.gob.es/) and by grants &#8220;Grupo de Referencia: E35_17R&#8221; and grant number &#8220;LMP220_21&#8221; from Diputaci&#243;n General de Arag&#243;n (DGA) (https://www.aragon.es/) to PF-S and RM-L.&#160;

NOTE: The composition of all culture media and buffers is specified in Table 1 and details related to all materials and reagents used in this protocol are listed in the Table of Materials.
1. Mitochondria isolation from cell culture
NOTE: The minimum volume of cells assayed has been ~30-50 &#181;L of packed cells (step 1.4). This can correspond approximately to at least two or three 100 mm cell culture plates or to one 150 mm plate at 80-90% of confluence, depending on the cell type (between 5 &#215; 106 and 107 cells in L929-derived cells or MDA-MB-468). Efficient cell breakage is a critical step.
<ol>
	<li>Grow adherent cells to approximately 80% confluence or suspended cells to an adequate density.</li>
	<li>Harvest cells by centrifugation at 500 &#215; g for 5 min (directly from the cell suspension in the case of non-adherent cells or after standard trypsinization with a trypsin-EDTA solution of 0.05% trypsin and 0.02% EDTA in 1x PBS, in the case of adherent cells).</li>
	<li>Wash cells 2x with cold 1x&#160;PBS and sediment them by centrifugation at 500 &#215; g for 5 min. 
	NOTE: From this point, all the steps must be performed at 4 &#176;C. Therefore, all the reagents must be cold and the tubes containing cells or mitochondria must be kept on ice.</li>
	<li>After the second centrifugation, discard the supernatant, estimate the cell pellet volume (cpv), and freeze the cells at -80 &#176;C for at least 15 min to facilitate membrane breaking in step 1.8. 
	NOTE: The protocol can be stopped at this point and the cells can be stored at -80 &#176;C for weeks. Usually, 10 or 15 mL graduated tubes with conical bottoms are appropriate.</li>
	<li>Let the cells thaw slowly on ice.</li>
	<li>Resuspend the packed cell pellet in a volume of hypotonic buffer equal to 7x the cpv (10 mM MOPS, 83 mM sucrose, pH 7.2) (e.g., 100 &#181;L of packed cells in 700 &#181;L of buffer). 
	NOTE: The volume must be enough to obtain efficient pops&#160;(see NOTE at step 1.8).</li>
	<li>Transfer the cell suspension into a Potter-Dounce homogenizer of the appropriate size and let the cells swell by incubating them in ice for 2 min. For example, for a volume of 700-800 &#181;L of cell suspension, a 1 mL capacity homogenizer is adequate.</li>
	<li>Break the cell membranes by performing eight to ten strokes in the homogenizer coupled to a motor-driven Teflon pestle rotating at 600 rpm. 
	NOTE: When making the strokes, it is important to create a vacuum that will increase the cell breakage efficiency along with the hypotonic and freezing actions for swelling and fragilizing the membranes, respectively. If it is well done, a &#34;pop&#34; sound will be heard when pulling down the homogenizer with a quick movement at each stroke.</li>
	<li>Add the same volume of hypertonic buffer to the cell suspension (7x the cpv) (30 mM MOPS, 250 mM sucrose, pH 7.2) to generate an isotonic environment.</li>
	<li>Transfer the homogenate into a 10-15 mL tube and centrifuge in a fixed rotor at 1,000 &#215; g for 5 min at 4 &#176;C. 
	NOTE: The original tube containing the unbroken cells can be used for this purpose.</li>
	<li>Transfer the supernatant, which contains the mitochondrial fraction, into 1.5 mL polypropylene tubes.</li>
	<li>OPTIONAL: To increase the yield of mitochondria, resuspend the pellet from step 1.10 in 7x the cell pellet volume of buffer A (10 mM Tris, 1 mM EDTA, 0.32 M sucrose, pH 7.4) by pipetting up and down and centrifuge at 1,000 &#215; g for 5 min at 4 &#176;C. Combine the new supernatant with the previous one before proceeding to step 1.13.</li>
	<li>Centrifuge in a microfuge at 16,000 &#215; g for 2 min at 4 &#176;C to collect the mitochondrial crude fraction.</li>
	<li>Discard the supernatant and resuspend each mitochondria-enriched pellet with 0.5 mL of buffer A combining the contents of two tubes into one and centrifuge under the same conditions as described in step 1.13. Repeat the same process until all the material is in only one tube.</li>
	<li>Discard the supernatant, resuspend the final pellet with 300 &#181;L of buffer A, and quantify mitochondrial protein concentration using the Bradford assay. Centrifuge again as before and proceed to section 3.</li>
</ol>
2. Isolation of mitochondria from small amounts of animal tissues
NOTE: The minimum amount of tissue to apply this protocol with a certain confidence depends on the cell type and its mitochondrial abundance but could be ~20-30 mg of tissue for most cases. The tissue samples can be fresh material or frozen samples. In the latter case, allow the samples to thaw in homogenization buffer placed on ice before starting the procedure.
<ol>
	<li>Weigh or estimate as closely as possible the amount (mg) of tissue.</li>
	<li>Cut the tissue with a pair of scissors and make 3-4 washes in homogenization buffer with the help of a strainer taking care to avoid losing the smaller pieces. 
	NOTE: This step is more important in tissues like skeletal muscle or heart than in the brain or softer tissues whose cells can be easily disaggregated directly by the homogenization step.</li>
	<li>Add fresh homogenization medium (4 mL per gram of liver, 10 mL per gram of heart, brown adipose tissue (BAT) or muscle, and 5 mL per gram of brain or kidney; see specific buffer composition for each case in Table 1).</li>
	<li>Transfer the pieces of tissue with buffer to the homogenizer and follow the optimal homogenization and mitochondria isolation protocol depending on the selected tissue.</li>
	<li>Isolation of mitochondria from liver, spleen, and kidney
	<ol>
		<li>Homogenize with four to six up-and-down strokes in the Elvehjem-Potter with a motor-driven Teflon pestle at 600 rpm.</li>
		<li>Transfer the homogenized tissue to a sterile centrifuge tube. Centrifuge in a swinging rotor at 1,000 &#215; g for 5 min at 4 &#176;C.</li>
		<li>Fill 1.5 mL polypropylene tubes with the supernatant obtained in the previous step. Centrifuge for 2 min at 16,000 &#215; g in a microfuge at 4 &#176;C and proceed hereafter as described before (steps 1.13 to 1.15)</li>
	</ol>
	</li>
	<li>Isolation of mitochondria from heart and muscle samples
	<ol>
		<li>Homogenize with six to eight strokes in the Elvehjem-Potter with a motor-driven Teflon pestle at 600 rpm. Transfer the homogenized tissue to a sterile centrifuge tube.</li>
		<li>Centrifuge in a swinging rotor at 1,000 &#215; g for 5 min at 4 &#176;C. Pour the supernatant into clean 1.5 mL polypropylene tubes. 
		NOTE: A second homogenization of the pellet obtained in step 2.6.2 can be performed with&#160;4-5 additional strokes and &#189; volume of homogenization buffer to increase the mitochondrial yield. The new supernatant (after centrifugation again at 1,000 &#215; g for 5 min at 4 &#176;C) can be combined with the previous one before proceeding to step 2.6.3. An alternative to increase the crude mitochondria yield is to dissociate heart and muscle tissue samples in trypsin solution before homogenization36,37. In this case, the trypsin has to be efficiently inactivated/removed before the mitochondrial membranes are solubilized with digitonin (step 3.2).</li>
		<li>Centrifuge at 16,000 &#215; g in a microfuge for 2 min at 4 &#176;C to obtain the crude mitochondrial fraction.</li>
		<li>Discard the supernatant and resuspend each mitochondria-enriched pellet with 0.5 mL of buffer AT combining the contents of two tubes into one and centrifuge under the same conditions as described in step 2.6.3. Repeat the same process until all the material is in only one tube.</li>
		<li>Discard the supernatant, resuspend the final pellet with 300 &#181;L of buffer A (no BSA), and quantify mitochondrial protein concentration using the Bradford assay. Centrifuge again as before and proceed to section 3.</li>
	</ol>
	</li>
	<li>Isolation of mitochondria from brain
	<ol>
		<li>Homogenize the pieces of the brain with 10-15 strokes using a Dounce-type glass homogenizer with a manually driven glass pestle. Transfer the homogenized tissue to a sterile centrifuge tube.</li>
		<li>Centrifuge in a swinging rotor at 1,000 &#215; g for 5 min at 4 &#176;C.</li>
		<li>Collect the supernatant into a clean centrifuge tube.</li>
		<li>Resuspend the pellet obtained in the previous centrifugation step in the same volume of medium AT used in the first homogenization. Re-homogenize the pellet by repeating the process described in step 2.7.1 using 5-10 passes and centrifuge the suspension in a swinging rotor at 1,000 &#215; g for 5 min at 4 &#176;C.</li>
		<li>Remove the supernatant and add it into the tube prepared in step 2.7.3. Centrifuge at 10,000 &#215; g in a fixed angle rotor for 10 min at 4 &#176;C to obtain the crude mitochondrial fraction.</li>
		<li>Discard the supernatant and resuspend the pellet into 1/2 the initial homogenization volume (step 2.7.1) of medium AT. Distribute the mitochondrial suspension into clean 1.5 mL polypropylene tubes and centrifuge at 16,000 &#215; g in a microfuge for 2 min at 4 &#176;C.</li>
		<li>Discard the supernatant and resuspend each mitochondria-enriched pellet with 0.5 mL of buffer AT combining the contents of two tubes into one and centrifuge under the same conditions as described in step 2.7.6. Repeat the same process until all the material is in only one tube.</li>
		<li>Discard the supernatant, resuspend the final pellet with 300 &#181;L of buffer A (no BSA), and quantify mitochondrial protein concentration using the Bradford assay. Centrifuge again as before and proceed to section 3.</li>
	</ol>
	</li>
	<li>Isolation of mitochondria from BAT
	<ol>
		<li>Homogenize with eight to ten up-and-down strokes in the Elvehjem-Potter with a motor-driven Teflon pestle at 600 rpm. Transfer the homogenized tissue to a sterile centrifuge tube.</li>
		<li>Centrifuge in a swinging rotor at 1,000 &#215; g for 5 min at 4 &#176;C.</li>
		<li>Fill 1.5 mL polypropylene tubes with the supernatant obtained in the previous step avoiding the upper fat layer. 
		&#8203;NOTE: A second homogenization of the pellet obtained in step 2.8.2 can be performed to increase the mitochondrial yield. The new supernatant (after centrifugation again at 1,000 &#215; g for 5 min at 4 &#176;C) can be combined with the previous one before proceeding to step 2.8.4.</li>
		<li>Centrifuge for 2 min at 16,000 &#215; g for 2 min in a microfuge at 4 &#176;C.</li>
		<li>Discard the supernatant and resuspend each mitochondria-enriched pellet with 0.5 mL of buffer AT2 combining the contents of two tubes into one and centrifuge under the same conditions as described in step 2.8.4. Repeat the same process until all the material is in only one tube.</li>
		<li>Discard the supernatant, resuspend the final pellet with 300 &#181;L of buffer A (no BSA), and quantify mitochondrial protein concentration using the Bradford assay. Centrifuge again as before and proceed to section 3.</li>
	</ol>
	</li>
</ol>
3. Preparation of samples for Blue Native analysis
<ol>
	<li>Resuspend the mitochondrial fractions (obtained from mammalian cell cultures or tissues) in BN sample buffer (50 mM NaCl, 50 mM Imidazole, 5 mM Aminocaproic acid, 4 mM PMSF) to obtain a protein concentration of around 10 mg/mL. See Table 2 for expected yields for the different types of samples.</li>
	<li>Solubilize mitochondrial membranes by adding digitonin (10% stock solution) to obtain a ratio of 4 g of digitonin/g of mitochondrial protein (4 &#181;L of digitonin 10% stock for 10 &#181;L of the mitochondrial suspension prepared in step 3.1). 
	NOTE: The detergent-to-protein ratio (g/g) must be optimized for each type of sample to obtain reproducible results; 2-8 g of digitonin/g of mitochondrial protein was found to be optimal for most tissues34,35.</li>
	<li>Mix by gently pipetting up and down. Incubate samples on ice for 5 min. 
	NOTE: After this step, the mitochondrial suspension should become clearer (shift from opaque to translucent); otherwise, it could indicate that the amount of detergent is insufficient for correct membrane solubilization.</li>
	<li>Centrifuge at full speed in a microfuge (20,000 &#215; g approx.) for 25 min at 4 &#176;C to remove insoluble material.</li>
	<li>Collect the supernatant in a fresh tube. Add to the supernatant a volume of 5% G-250 (Coomassie Blue G-250 5% in 0.75 M aminocaproic acid) equivalent to 1/3 of the initial resuspension volume (step 3.1, this would correspond to a final proportion of 1.6 g of Coomassie/g of protein) and mix by pipetting.</li>
	<li>Keep on ice before loading on the gel or freeze aliquots at -80 &#176;C.</li>
</ol>
4. Blue Native gel electrophoresis
NOTE: The electrophoresis is performed in the cold room (4-8 &#176;C). If there is no cold room facility, a cooling block can be introduced in the electrophoresis tank. Commercial 3-13% native polyacrylamide gels29 are used, but homemade gels of the desired gradient concentrations can also be used38,39.
<ol>
	<li>Load the upper and lower chambers with the cold cathode A and anode buffers, respectively. Alternatively, load the samples in the wells before carefully filling the upper chamber with cathode buffer A. 
	NOTE: Commercial electrophoresis buffers are used in our laboratory, but they can be prepared as indicated in Table 1.</li>
	<li>Load between 30 and 100 &#181;g of mitochondrial protein. 
	NOTE: Usually, 30-50 &#181;g of protein (for WB), or 40-100 &#181;g of protein (for IGA) are loaded per well in a 10 lane gel (0.5 x 0.15 cm wells). Cathode A buffer contains Coomassie Blue G-250 and has an intense blue color that makes it difficult to see the gel wells for sample loading. It is convenient to mark the center of each well, with a red marker, for example, to help in placing the pipette tip during this step. Native molecular weight markers can be useful when setting up the BN-PAGE technique to confirm that the gradient gels are properly formed and that the complexes and SCs pattern is the correct one.</li>
	<li>Run at 80-100 V for 25-30 min and then at 160-180 V, limiting the current to 12 mA/gel, until the dye reaches the bottom of the gel (~125-165 min in total).</li>
	<li>If in-gel activity assays (IGAs) are to be performed, substitute the cathode buffer A with cathode buffer B when the dye front is in the middle of the gel (~1 h after the beginning of the run). 
	NOTE: Although the gels can be documented without any staining just after the run since some bands are visible (mainly complex V, which can be considered an &#34;internal&#34; maker with a MW of ~600 kDa) due to their binding to G-250 dye, usually, SCs and individual complexes will not be visible without staining or IGA assays. The gels to be used in IGA assays or for WB should not be stained or fixed.</li>
	<li>Disassemble the gel from the cassette and continue with Coomassie blue staining (section 5), IGA analysis (section 6), or WB immunodetection (section 7).</li>
</ol>
5. Gel staining
<ol>
	<li>Stain the gel for 10-15 min at room temperature (RT) using Coomassie blue dye solutions (Coomassie dye R-250 at 0.25% in 40% methanol, 10% acetic acid; stain for 10-15 min).</li>
	<li>Destain with several washes (typically 4-5 x 15 min) in 40% methanol plus 10% acetic acid at RT.</li>
	<li>Document the gel.</li>
</ol>
6. In gel-activity (IGA) assays
<ol>
	<li>Prepare IGA solutions for the analysis of the different respiratory complexes before the end of the electrophoresis and maintain them in the dark (by using dark-colored plastic boxes or covering them with aluminum foil, for example). The composition of each buffer is detailed in Table 1.</li>
	<li>Place the gel or the lanes to be used in a plastic box as small as possible to accommodate it.</li>
	<li>Add enough volume of the appropriate solution to cover the gel (usually 5 or 10 mL for 5 or 10 gel lanes, respectively) and incubate at RT with gentle shaking (60-80 rpm) and away from light. 
	NOTE: The time of incubation depends on the complex to be analyzed and on the nature and amount of sample loaded; CI and CIV are the easiest and fastest to give results. Thus, CI activity will usually start to become visible after a few minutes, while CII and CIV need ~30 min to be observed and CV ~1.5-2 h. The reaction can continue for hours in all cases and the rate of signal intensification can be reduced by moving the incubation to a cold room (4-6 &#176;C), for example, for an overnight (ON) incubation. CIII activity works only for clear native electrophoresis (CN), not for BN34.</li>
	<li>When the appropriate bands have developed, stop the reactions by removing the assay solutions, washing 2x with distilled water, and fixing the gel with 40% methanol plus 10% acetic acid (except for CV which is fixed only with methanol) for 30 min.
	<ol>
		<li>To analyze CIV activity after CI detection by IGA, wash the gel 2x with distilled water, document CI activity, incubate the gel (without fixing it!) with 50 mM potassium phosphate buffer (pH 7.2) 2x for 30 min, and then with the complete CIV reaction buffer until the complex IV bands appear (usually by incubating ON at RT or 4 &#176;C).</li>
	</ol>
	</li>
	<li>Document the gels. 
	NOTE: For ATPase activity, as the developed bands are white, when documenting the gel, place it on a dark background so that the clear bands would be visible.</li>
</ol>
7. Western blot analysis
<ol>
	<li>Prepare transfer buffer according to Table 1 and keep at 4 &#176;C until the end of the electrophoresis.</li>
	<li>Place the gel in a tray and add transfer buffer. Incubate at RT for 10-15 min.</li>
	<li>Cut a piece of PVDF membrane of the same size as the gel and activate it in methanol for 10 s under agitation. Wash several times with distilled water and add transfer buffer. Incubate at RT for 10-15 min with gentle shaking.</li>
	<li>Prepare the transfer sandwich, from bottom to top, avoiding bubbles between the gel and the membrane, in the following order: black side of cassette-sponge-blotting paper-gel-membrane-blotting paper- sponge-clear side of cassette.</li>
	<li>Close and lock the cassette and put the sandwich in the transfer tank in the correct orientation for transfer (black side towards the negative pole).</li>
	<li>Fill the transfer tank with transfer buffer, add a magnetic stirrer to agitate the buffer, and connect the power supply at 80 V for 2 h or at 100 V for 1 h. Perform the transfer between 4 and 8 &#176;C (for example in the cold room) and with a cooling block in the transfer system.</li>
	<li>Retrieve the membrane and continue with a standard western blot protocol, using specific antibodies for the different respiratory complexes to be detected29.</li>
</ol>

Mitochondrial Fractionation in Small Tissue Samples for Supercomplex Analysis

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R., Flores-Garcia, M., Pedraza-Chaverri, J., Zazueta, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alteration+of+mitochondrial+supercomplexes+assembly+in+metabolic+diseases.">Alteration of mitochondrial supercomplexes assembly in metabolic diseases.</a> Biochim Biophys Acta Mol Basis Dis. 1866 (12), 165935 (2020).</li><li>Gonzalez-Rodriguez, P., 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=Disruption+of+mitochondrial+complex+I+induces+progressive+parkinsonism.">Disruption of mitochondrial complex I induces progressive parkinsonism.</a> Nature. 599 (7886), 650-656 (2021).</li><li>Novack, G. V., Galeano, P., Castano, E. M., Morelli, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+supercomplexes:+physiological+organization+and+dysregulation+in+age-related+neurodegenerative+disorders.">Mitochondrial supercomplexes: physiological organization and dysregulation in age-related neurodegenerative disorders.</a> Front Endocrinol (Lausanne). 11, 600 (2020).</li><li>Ramirez-Camacho, I., Flores-Herrera, O., Zazueta, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relevance+of+the+supramolecular+arrangements+of+the+respiratory+chain+complexes+in+human+diseases+and+aging.">The relevance of the supramolecular arrangements of the respiratory chain complexes in human diseases and aging.</a> Mitochondrion. 47, 266-272 (2019).</li><li>Hollinshead, K. E. 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=Respiratory+Supercomplexes+Promote+Mitochondrial+Efficiency+and+Growth+in+Severely+Hypoxic+Pancreatic+Cancer.">Respiratory Supercomplexes Promote Mitochondrial Efficiency and Growth in Severely Hypoxic Pancreatic Cancer.</a> Cell Rep. 33 (1), 108231 (2020).</li><li>Ikeda, K., 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=Mitochondrial+supercomplex+assembly+promotes+breast+and+endometrial+tumorigenesis+by+metabolic+alterations+and+enhanced+hypoxia+tolerance.">Mitochondrial supercomplex assembly promotes breast and endometrial tumorigenesis by metabolic alterations and enhanced hypoxia tolerance.</a> Nat Commun. 10 (1), 4108 (2019).</li><li>Kamada, S., Takeiwa, T., Ikeda, K., Horie, K., Inoue, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+roles+of+COX7RP+and+mitochondrial+oxidative+phosphorylation+in+breast+cancer.">Emerging roles of COX7RP and mitochondrial oxidative phosphorylation in breast cancer.</a> Front Cell Dev Biol. 10, 717881 (2022).</li><li>Marco-Brualla, J., 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=Mutations+in+the+ND2+subunit+of+mitochondrial+complex+I+are+sufficient+to+confer+increased+tumorigenic+and+metastatic+potential+to+cancer+cells.">Mutations in the ND2 subunit of mitochondrial complex I are sufficient to confer increased tumorigenic and metastatic potential to cancer cells.</a> Cancers (Basel). 11 (7), 1027 (2019).</li><li>Moreno-Loshuertos, 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=How+hot+can+mitochondria+be?+Incubation+at+temperatures+above+43+degrees+C+induces+the+degradation+of+respiratory+complexes+and+supercomplexes+in+intact+cells+and+isolated+mitochondria.">How hot can mitochondria be? Incubation at temperatures above 43 degrees C induces the degradation of respiratory complexes and supercomplexes in intact cells and isolated mitochondria.</a> Mitochondrion. 69, 83-94 (2023).</li><li>Vonck, J., Schafer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supramolecular+organization+of+protein+complexes+in+the+mitochondrial+inner+membrane.">Supramolecular organization of protein complexes in the mitochondrial inner membrane.</a> Biochim Biophys Acta. 1793 (1), 117-124 (2009).</li><li>Althoff, T., Mills, D. J., Popot, J. L., Kuhlbrandt, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arrangement+of+electron+transport+chain+components+in+bovine+mitochondrial+supercomplex+I1III2IV1.">Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1.</a> EMBO J. 30 (22), 4652-4664 (2011).</li><li>Cogliati, S., 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=Mechanism+of+super-assembly+of+respiratory+complexes+III+and+IV.">Mechanism of super-assembly of respiratory complexes III and IV.</a> Nature. 539 (7630), 579-582 (2016).</li><li>Gonzalez-Franquesa, A., 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=Mass-spectrometry-based+proteomics+reveals+mitochondrial+supercomplexome+plasticity.">Mass-spectrometry-based proteomics reveals mitochondrial supercomplexome plasticity.</a> Cell Rep. 35 (8), 109180 (2021).</li><li>Wittig, I., Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+and+applications+of+blue-native+and+clear-native+electrophoresis.">Features and applications of blue-native and clear-native electrophoresis.</a> Proteomics. 8 (19), 3974-3990 (2008).</li><li>Wittig, I., Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Native+electrophoretic+techniques+to+identify+protein-protein+interactions.">Native electrophoretic techniques to identify protein-protein interactions.</a> Proteomics. 9 (23), 5214-5223 (2009).</li><li>Garcia-Cazarin, M. L., Snider, N. N., Andrade, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+isolation+from+skeletal+muscle.">Mitochondrial isolation from skeletal muscle.</a> J Vis Exp. (49), e2452 (2011).</li><li>Lai, N., 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=Isolation+of+mitochondrial+subpopulations+from+skeletal+muscle:+Optimizing+recovery+and+preserving+integrity.">Isolation of mitochondrial subpopulations from skeletal muscle: Optimizing recovery and preserving integrity.</a> Acta Physiol (Oxf). 225 (2), e13182 (2019).</li><li>Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Native+electrophoresis+for+isolation+of+mitochondrial+oxidative+phosphorylation+protein+complexes.">Native electrophoresis for isolation of mitochondrial oxidative phosphorylation protein complexes.</a> Methods Enzymol. 260, 190-202 (1995).</li><li>Wittig, I., Braun, H. P., Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+PAGE.">Blue native PAGE.</a> Nat Protoc. 1 (1), 418-428 (2006).</li><li>Chomyn, A., 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=Platelet-mediated+transformation+of+mtDNA-less+human+cells:+analysis+of+phenotypic+variability+among+clones+from+normal+individuals--and+complementation+behavior+of+the+tRNALys+mutation+causing+myoclonic+epilepsy+and+ragged+red+fibers.">Platelet-mediated transformation of mtDNA-less human cells: analysis of phenotypic variability among clones from normal individuals--and complementation behavior of the tRNALys mutation causing myoclonic epilepsy and ragged red fibers.</a> Am J Hum Genet. 54 (6), 966-974 (1994).</li><li>Moreno-Loshuertos, 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=Differences+in+reactive+oxygen+species+production+explain+the+phenotypes+associated+with+common+mouse+mitochondrial+DNA+variants.">Differences in reactive oxygen species production explain the phenotypes associated with common mouse mitochondrial DNA variants.</a> Nat Genet. 38 (11), 1261-1268 (2006).</li><li>Fern&#225;ndez-Vizarra, E., Fern&#225;ndez-Silva, P., Enr&#237;quez, J. A., Celis, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Biology (Third Edition). , 69-77 (2006).</li><li>Cogliati, S., Herranz, F., Ruiz-Cabello, J., Enr&#237;quez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digitonin+concentration+is+determinant+for+mitochondrial+supercomplexes+analysis+by+BlueNative+page.">Digitonin concentration is determinant for mitochondrial supercomplexes analysis by BlueNative page.</a> Biochim Biophys Acta Bioenerg. 1862 (1), 148332 (2021).</li></ol>

The authors declare no conflicts of interest.

The methodological adaptations introduced in the protocols described&#160;here are intended to avoid losses and increase the yield while maintaining mitochondrial complex activities (which is crucial when the availability of enough amounts of samples is compromised) and reproduce the tissue&#39;s or cell line&#39;s expected pattern of SCs (see Figure 2C). With this purpose and since a high mitochondrial purity is not required to properly detect the SCs, the number of steps, times, and volume...

Supercomplexes (SCs) are supramolecular associations between individual respiratory chain complexes1,2. Since the initial identification of SCs and the description of their composition by the group of Sch&#228;gger2,3, later confirmed by other groups, it was established that they contain respiratory complexes I, III, and IV (CI, CIII, and CIV, respectively) in different stoichiometries. Two main populations of SCs can be defined, those containing CI (and either CIII alone or CIII and CIV) and with very high molecular weight (MW, starting ~1.5 MDa for the smaller SC: CI + CIII2) and those containing CIII and CIV but not CI, with much smaller size (such as CIII2 + CIV with ~680 kDa). These SCs coexist in the inner mitochondrial membrane with free complexes, also in different proportions. Thus, while CI is mostly found in its associated forms (that is, in SCs: ~80% in bovine heart and more than 90% in many human cell types)3, CIV is very abundant in its free form (more than 80% in bovine heart), with CIII showing a more balanced distribution (~40% in its more abundant free form, as a dimer, in bovine heart).
While their existence is now generally accepted, their precise role is still under debate4,5,6,7,8,9,10. According to the plasticity model, different proportions of SCs and individual complexes can exist depending on the cell type or the metabolic status1,7,11. This dynamic nature of the assembly would allow cells to regulate the use of available fuels and the efficiency of the oxidative phosphorylation (OXPHOS) system in response to environmental changes4,5,7. SCs could also contribute to control the reactive oxygen species generation rate and participate in the stabilization and turnover of individual complexes4,12,13,14. Modifications of the SC assembly status have been described in association with different physiological and pathological situations15,16 and with the aging process17.
Thus, changes in the SC patterns have been described in yeast depending on the carbon source used for growth2&#160;and in cultured mammalian cells when glucose is substituted by galactose4. Modifications have also been reported in mouse liver after fasting8 and in astrocytes when mitochondrial fatty acid oxidation is blocked18. In addition, a decrease or alterations in SCs and OXPHOS have been found in Barth syndrome19, heart failure20, several metabolic21 and neurological22,23,24 disorders, and different tumors25,26,27,28. Whether these alterations in SC assembly and levels are a primary cause or represent secondary effects in these pathological situations is still under investigation15,16. Different methodologies can give information about the assembly and function of SCs; these include activity measurements8,29, ultrastructural analysis30,31, and proteomics32,33. A useful alternative that is increasingly being employed and is the starting point for some of the previously mentioned methodologies is the direct determination of SC assembly status by Blue native (BN) electrophoresis developed for this purpose by Sch&#228;gger&#39;s group34,35.
This approach requires reproducible and efficient procedures to obtain and solubilize mitochondrial membranes and can be complemented by other techniques such as in-gel activity analysis (IGA), second-dimension electrophoresis, and western blot (WB). A limitation in the studies on SC dynamics by BN electrophoresis can be the amount of starting cells or tissue samples. We present a series of protocols for the analysis of SC assembly and function, adapted from Sch&#228;gger&#39;s group methods, that can be applied to fresh or frozen cell or tissue samples starting from as little as 20 mg of tissue.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetic acid</td>
			<td>PanReac</td>
			<td>131008</td>
			<td></td>
		</tr>
		<tr>
			<td>Aminocaproic acid</td>
			<td>Fluka Analytical</td>
			<td>7260</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma-Aldrich</td>
			<td>A2383</td>
			<td></td>
		</tr>
		<tr>
			<td>Bis Tris</td>
			<td>Acrons Organics</td>
			<td>327721000</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford assay</td>
			<td>Biorad</td>
			<td>5000002</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Blue G-250</td>
			<td>Serva</td>
			<td>17524</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Blue R-250</td>
			<td>Merck</td>
			<td>1125530025</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytochrome c</td>
			<td>Sigma-Aldrich</td>
			<td>C2506</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamino&#160; benzidine (DAB)</td>
			<td>Sigma-Aldrich</td>
			<td>D5637</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitonin</td>
			<td>Sigma-Aldrich</td>
			<td>D5628</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>PanReac</td>
			<td>131669</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Fatty acids free BSA</td>
			<td>Roche</td>
			<td>10775835001</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>PanReac</td>
			<td>A1067</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer Teflon pestle</td>
			<td>Deltalab</td>
			<td>196102</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I2399</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>PanReac</td>
			<td>121512</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>PanReac</td>
			<td>121509</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Sigma-Aldrich</td>
			<td>M4125</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Labkem</td>
			<td>MTOL-P0P</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>PanReac</td>
			<td>131404</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Trans-Blot Cell</td>
			<td>BioRad</td>
			<td>1703930</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma-Aldrich</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr>
			<td>MTCO1 Monoclonal Antibody</td>
			<td>Invitrogen</td>
			<td>459600</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>NADH</td>
			<td>Roche</td>
			<td>10107735001</td>
			<td></td>
		</tr>
		<tr>
			<td>NativePAGE 3 to 12% Mini Protein Gels</td>
			<td>Invitrogen</td>
			<td>BN1001BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>NativePAGE Cathode Buffer Additive (20x)</td>
			<td>Invitrogen</td>
			<td>BN2002</td>
			<td></td>
		</tr>
		<tr>
			<td>NativePAGE Running Buffer (20x)&#160;</td>
			<td>Invitrogen</td>
			<td>BN2001</td>
			<td></td>
		</tr>
		<tr>
			<td>NDUFA9 Monoclonal Antibody</td>
			<td>Invitrogen</td>
			<td>459100</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitroblue tetrazolium salt (NBT)</td>
			<td>Sigma-Aldrich</td>
			<td>N6876</td>
			<td></td>
		</tr>
		<tr>
			<td>Pb(NO3)2</td>
			<td>Sigma-Aldrich</td>
			<td>228621</td>
			<td></td>
		</tr>
		<tr>
			<td>PDVF Membrane</td>
			<td>Amersham</td>
			<td>10600023</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenazine methasulfate (PMS)</td>
			<td>Sigma-Aldrich</td>
			<td>P9625</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce ECL Substrate</td>
			<td>Thermo Scientific</td>
			<td>32106</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Merck</td>
			<td>PMSF-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>SDHA Monoclonal Antibody</td>
			<td>Invitrogen</td>
			<td>459200</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium succinate</td>
			<td>Sigma-Aldrich</td>
			<td>S2378</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin/penicillin</td>
			<td>PAN biotech</td>
			<td>P06-07100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S3089</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>PanReac</td>
			<td>A2264</td>
			<td></td>
		</tr>
		<tr>
			<td>UQCRC1 Monoclonal Antibody</td>
			<td>Invitrogen</td>
			<td>459140</td>
			<td></td>
		</tr>
		<tr>
			<td>XCell&#160;SureLock Mini-Cell</td>
			<td>Invitrogen</td>
			<td>&#160;EI0001</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The yields of mitochondria obtained following the above-described protocols vary depending on several factors such as the cell line or tissue type, the nature of the samples (i.e., if fresh or frozen tissues are used), or the efficiency of the homogenization process. Expected yields of mitochondria from different cell lines and tissues are collected in Table 2. Once the mitochondrial fractions have been obtained, the next step is the analysis of respiratory SCs pattern, which is performed after the crude...

Read this Scientific Article Protocol about Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples at JoVE.com

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.

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 ...

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JoVE Journal

Biochemistry

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

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

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.

We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

Isolation of Tissue Extracellular Vesicles from the Liver

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in cell-to-cell communication by shuttling bioactive molecules such as RNA or protein from one cell to another. Most studies of EVs have been performed in cell culture models or in EVs isolated from body fluids. There is emerging interest in the isolation of EVs from tissues to study their contribution to physiological processes and how they are altered in disease. The isolation of EVs with sufficient yield from tissues is technically challenging because of the need for tissue dissociation without cellular damage. This method describes a procedure for the isolation of EVs from mouse liver tissue. The method involves a two-step process starting with in situ collagenase digestion followed by differential ultra-centrifugation. Tissue perfusion using collagenase provides an advantage over mechanical cutting or homogenization of liver tissue due to its increased yield of obtained EVs. The use of this two-step process to isolate EVs from the liver will be useful for the study of tissue EVs.

This is a protocol to isolate tissue extracellular vesicles (EVs) from the liver. The protocol describes a two-step process involving collagenase perfusion followed by differential ultracentrifugation to isolate liver tissue EVs.

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Measuring Mitochondrial Electron Transfer Complexes in Previously Frozen Cardiac Tissue from the Offspring of Sow: A Model to Assess Exercise-Induced Mitochondrial Bioenergetics Changes

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis proposed and tested was that a regular maternal exercise of a sow during pregnancy would increase the mitochondrial efficiency of offspring heart bioenergetics. This hypothesis was tested by isolating mitochondria using a mild-isolation procedure to assess mitochondrial ETC and supercomplex profiles. The procedure described here allowed for the processing of previously frozen archived heart tissues and eliminated the necessity of fresh mitochondria preparation for the assessment of mitochondrial ETC complexes, supercomplexes, and ETC complex activity profiles. This protocol describes the optimal ETC protein complex measurement in multiplexed antibody-based immunoblotting and super complex assessment using blue-native gel electrophoresis.

Preparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria.

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis ...

Measuring Ubiquitylated, SUMOylated, and ADP-Ribosylated DNA-Protein Crosslinks

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, methyltransferases, etc.) malfunctioning, or exogenous agents such as chemotherapeutics and crosslinking agents. Once DPCs are induced, several types of post-translational modifications (PTMs) are promptly conjugated to them as early response mechanisms. It has been shown that DPCs can be modified by ubiquitin, small ubiquitin-like modifier (SUMO), and poly-ADP-ribose, which prime the substrates to signal their respective designated repair enzymes and, in some cases, coordinate the repair in sequential manners. As PTMs transpire quickly and are highly reversible, it has been challenging to isolate and detect PTM-conjugated DPCs that usually remain at low levels. Presented here is an immunoassay to purify and quantitatively detect ubiquitylated, SUMOylated, and ADP-ribosylated DPCs (drug-induced topoisomerase DPCs and aldehyde-induced non-specific DPCs) in vivo. This assay is derived from the RADAR (rapid approach to DNA adduct recovery) assay that is used for the isolation of genomic DNA containing DPCs by ethanol precipitation. Following normalization and nuclease digestion, PTMs of DPCs, including ubiquitylation, SUMOylation, and ADP-ribosylation, are detected by immunoblotting using their corresponding antibodies. This robust assay can be utilized to identify and characterize novel molecular mechanisms that repair enzymatic and non-enzymatic DPCs&#160;and has the potential to discover small molecule inhibitors targeting specific factors that regulate PTMs to repair DPCs.

Author Spotlight: Quantitative Detection of DNA Protein Crosslinks and Their Post-Translational Modifications  

The present protocol highlights a modified method to detect and quantify DNA-protein crosslinks (DPCs) and their post-translational modifications (PTMs), including ubiquitylation, SUMOylation, and ADP-ribosylation induced by topoisomerase inhibitors and by formaldehyde, thereby allowing the study of the formation and repair of DPCs and their PTMs.

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, ...

Isolation of Mitochondrial Contact Sites

An Improved Method to Isolate Mitochondrial Contact Sites

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the synthesis of iron-sulfur clusters, lipids, or proteins, Ca2+ buffering, and the induction of apoptosis. Likewise, mitochondrial dysfunction results in severe human diseases such as cancer, diabetes, and neurodegeneration. In order to perform these functions, mitochondria have to communicate with the rest of the cell across their envelope, which consists of two membranes. Therefore, these two membranes have to interact constantly. Proteinaceous contact sites between the mitochondrial inner and outer membranes are essential in this respect. So far, several contact sites have been identified. In the method described here, Saccharomyces cerevisiae mitochondria are used to isolate contact sites and, thus, identify candidates that qualify for contact site proteins. We used this method to identify the mitochondrial contact site and cristae organizing system (MICOS) complex, one of the major contact site-forming complexes in the mitochondrial inner membrane, which is conserved from yeast to humans. Recently, we further improved this method to identify a novel contact site consisting of Cqd1 and the Por1-Om14 complex.

Author Spotlight: Unveiling Mitochondrial Contact Sites and Architectural Insights 

Mitochondrial contact sites are protein complexes that interact with mitochondrial inner and outer membrane proteins. These sites are essential for the communication between the mitochondrial membranes and, thus, between the cytosol and the mitochondrial matrix. Here, we describe a method to identify candidates qualifying for this specific class of proteins.

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the ...

Enhancing EV Isolation for Precise Biofluid Analysis

Chemical Affinity-Based Isolation of Extracellular Vesicles from Biofluids for Proteomics and Phosphoproteomics Analysis

Extracellular vesicles (EVs) from biofluids have recently gained significant attention in the field of liquid biopsy. Released by almost every type of cell, they provide a real-time snapshot of host cells and contain a wealth of molecular information, including proteins, in particular those with post-translational modifications (PTMs) such as phosphorylation, as the main player of cellular functions and disease onset and progression. However, the isolation of EVs from biofluids remains challenging due to low yields and impurities from current EV isolation methods, making the downstream analysis of EV cargo, such as EV phosphoproteins, difficult. Here, we describe a rapid and effective EV isolation method based on functionalized magnetic beads for EV isolation from biofluids such as human urine and downstream proteomics and phosphoproteomics analysis following EV isolation. The protocol enabled a high recovery yield of urinary EVs and sensitive profiles of EV proteome and phosphoproteome. Furthermore, the versatility of this protocol and relevant technical considerations are also addressed here.

Author Spotlight: Advancing EVtrap for High-Throughput Proteomics in Disease Biomarker Discovery 

The present protocol provides detailed descriptions for the efficient isolation of urinary extracellular vesicles utilizing functionalized magnetic beads. Moreover, it encompasses subsequent analyses, including western blotting, proteomics, and phosphoproteomics.

Extracellular vesicles (EVs) from biofluids have recently gained significant attention in the field of liquid biopsy. Released by almost every type of ...