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., 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 volumes have been reduced whenever this was possible.
Thus, after homogenization and removal of nuclei and unbroken cells by low-speed centrifugation, the supernatants containing the mitochondria are transferred to 1.5 mL polypropylene tubes, and the subsequent centrifugations to obtain the crude mitochondrial fractions are performed in a microfuge by increasing the speed and reducing the times and volumes with respect to conventional protocols. This allows the processing of several samples in a relatively short time: a close estimation is that 10 samples can be processed in ~2-2.5 h (from the start of the homogenization (protocol step 1.6. or equivalent for tissues) to the obtention of the SCs fraction ready to be loaded in a gel (protocol step 3.7)&#160;and minimizes the losses while keeping activities. Another change with respect to the original Sch&#228;gger&#39;s group protocols is to substitute, after membrane solubilization for the obtention of the SCs fraction, an ultracentrifugation step by a simple microfuge centrifugation (protocol step 3.4).
There are some critical steps in the procedure. First, the efficiency of cell breakage, especially in the case of cultured cells (see note after protocol step 1.8) and particularly in the case of small cells. Tissues and cell lines present a variable difficulty degree in cell breakage: for example, in the heart and muscle, this step is usually less efficient than in the liver or spleen, but it is compensated in yield because the former have a higher mitochondrial content. One alternative to improve mitochondrial yield when isolated from muscle or heart would be the treatment of samples with trypsin before homogenization. The brain is another tissue that presents low efficiency in cell breakage because of the manual homogenization, which is milder and less efficient and probably also because of the higher variation in cell types in this tissue, as it has been reported previously 42. This can be partially compensated by a second homogenization step as suggested in our protocol (step 2.7.4). On the other hand, if cell breakage is excessive, the final sample containing SCs could be contaminated with nuclear DNA fragments that may interfere with the electrophoretic running. This problem is shown as an increase in the viscosity of the sample when digitonin is added to solubilize the mitochondrial membranes and can be solved by treatment with DNAse I.
Second, if the detergent-to-protein ratio is too low, that would result in less efficient membrane solubilization and the possibility of aggregate formation. This could show in the gels as poor resolution in high molecular weight SCs (see Figure 4) or as CIV giving multiple bands corresponding to oligomers43. If the ratio is too high, it could lead to disaggregation of SCs (mainly CI-SCs). Third, it is important to avoid heating samples during the whole procedure (see Figure 4) and avoid repeated cycles of freezing-thawing since they may affect SC assembly status.
In Table 2, a summary of expected yields is presented for several cell types and tissue samples. The values are approximate and will depend on the cell breakage efficiency and the nature of the source. In the case of tissues, if the starting material is frozen it tends to give a higher yield than with fresh samples. An estimation of the number of lanes (considering a load between 50 and 60 &#181;g of mitochondrial protein per lane) that can be loaded is presented.
The protocols presented here cover a variety of tissues and cell types with small variations, they can be used with fresh or frozen samples and require simple equipment and a relatively low cost, not involving the use of expensive purification kits or gradients. However, they may need some adaptations to be used with particular cell types or samples, as well as when specific mitochondrial populations, such as brain synaptic mitochondria or muscle subsarcolemmal mitochondria, for example, are the object of analysis.
Since the precise physiological roles as well as the potential involvement of SCs in pathological situations remains to be defined, the analysis of their dynamics and the factors affecting their assembly, stability, and function is needed. Our protocols can represent valuable tools that allow&#160;to obtain reproducible results on the SCs&#160;patterns with small sample sizes and from a variety of cell types and tissues, particularly when multiple experimental conditions need to be compared29.

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 mitochondrial sample solubilization and electrophoretic separation by BN-PAGE followed by IGA-analysis or WB immunodetection. Figure 1 shows the aspect of unstained gel lanes just after the run and the pattern of bands after standard Coomassie staining of a cultured cell line and a mouse liver mitochondrial sample.
In Figure 2A,B, clear differences between the SC assembly patterns in human and mouse cells after using IGA assays can be observed. Thus, free complex I is observed in mouse cells whereas it is not detectable in human mitochondria. Complex IV-IGA pattern is very similar in both human cell lines (Figure 2B) while presenting differences between the two mouse cell lines analyzed in Figure 2A. These differences are due to the fact that BL6 cells express a mutant variant of SCAF18.
The SC patterns, obtained with the protocol for mitochondrial isolation described here to work with small samples, are maintained with respect to the &#34;conventional&#34; protocols used for larger samples. This can be seen in Figure 2C, by comparing lanes 1 and 6 (obtained after mitochondrial isolation using a conventional protocol from 3 g of liver and 1.1 g of BAT) with, respectively, lanes 2 and 5 (obtained using the protocols here presented from around 0.1 g of both tissues). As proposed in the plasticity model, the relative proportion of respiratory complexes and SCs varies depending on the cell type and the metabolic state. As shown in Figure 2C,D, the different tissues show different SC assembly patterns. Thus, brain mitochondria show very low levels of free complex I and a higher proportion of SCs when compared with the other tissues, and BAT is characterized by its low levels of CV (Figure 2A) and high amounts of SC III + IV (Figure 2D). Heart mitochondria present high levels of SCs and CV. CV assembly patterns are very similar between the cultured cell line and a mouse liver mitochondrial sample (Figure 2E).
SC assembly can also be analyzed by WB as shown in Figure 3 for two samples obtained from spleen control and tumor cells. In this case, 30-50 &#181;g of mitochondrial protein are usually enough to detect the different complexes and SCs with the specific antibodies. Figure 4 shows some of the main pitfalls that can happen during the application of these protocols. Wrong gel gradients can produce abnormal and undesired migration patterns as in Figure 4A, lane 1, where the SCs remain close to the well in the upper part of the lane due to a higher-than-normal acrylamide concentration in the gel. Incubation of the samples, even for relatively short times, at high temperatures (here 37 &#176;C and 40 &#176;C for 10 min) causes degradation of SCs and CI (Figure 4A lanes 2-4). Likewise, the detergent-to-protein ratio is critical for good results and adequate gel resolution. Below a ratio of 2 g of digitonin/g of protein, the band resolution is progressively reduced and at the lower concentration (0.5 g/g) only a smear in the area corresponding to SCs is visible (Figure 4B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66771/66771fig01.jpg" /> 
Figure 1: Pattern of mitochondrial complexes and supercomplexes after BNGE separation of digitonin-permeabilized mitochondria from a culture cell line (L929Balbc) and mouse liver. The lanes on the left (1 and 2) represent the aspect of the unstained gel just after the run, where only CV is detectable, while the lanes on the right (3 and 4) show the pattern of bands after Coomassie staining. Abbreviations: BNGE = Blue Native gel electrophoresis; CV = complex V; SCs = supercomplexes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66771/66771fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66771/66771fig02.jpg" /> 
Figure 2: IGA-analysis of mitochondria isolated from different cell lines and mouse tissues. (A,B) In-gel activity of the indicated complexes in mitochondria isolated from (A) mouse or (B) human cell lines. L929Balbc is a transmitochondrial cell line generated in our laboratory by transferring mitochondria from Balb/cJ mouse platelets to &#961;&#176;L929neo cells as described previously40,41. BL6 fibroblasts are an immortalized cell line generated in our lab from an ear biopsy of a BL6 mouse. (C) CI-IGA patterns from mouse mitochondria isolated using either a conventional method starting with 3 g of liver (lane 1) and with 1.1 g of BAT (lane 6) or the protocol presented here and starting with around 0.1 g of tissue in all cases (lane 2, liver; lane 3, heart; lane 4, brain, and lane 5 BAT). (D) CIV-IGA patterns (performed after CI-IGA shown in panel 2C, lanes 2-5) in mouse liver, heart, brain, and BAT mitochondria. (E) CV-IGA activity analyzed in mouse cultured cells and liver. Around 60-75 &#181;g of mitochondrial protein per lane were loaded. Abbreviations: IGA = in-gel activity; SCs = supercomplexes; CI = complex I; BAT = brown adipose tissue. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66771/66771fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66771/66771fig03.jpg" /> 
Figure 3: WB immunodetection of the different mitochondrial complexes and supercomplexes in spleen samples. After Blue Native PAGE separation of mitochondrial complexes and SCs obtained from spleen control (Spleen M9) and tumor (Tumor M9) samples, they were transferred to a PVDF membrane and sequentially hybridized with antibodies that recognize the indicated CI, CIII, CIV, and CII subunits (panels A-D, respectively). Around 50 &#181;g of mitochondrial protein were loaded per lane. Asterisks indicate the signal remaining from a previous western blot. Abbreviations: SC = supercomplex; CI = complex I; CIII = complex III; CIV = complex IV; CII = complex II. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66771/66771fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66771/66771fig04v2.jpg" /> 
Figure 4: Pitfalls in SC analysis by Blue-Native PAGE. CI IGA-analysis of mitochondria isolated from (A) fresh mouse liver and (B) frozen rat liver under different solubilization conditions. (A) Lane 1, wrong gel gradient concentration (8-13% instead of the normal 3-13%). Lanes 2-4, samples incubated for 10 min on ice or at 37 &#176;C and 40 &#176;C before loading, respectively. (B) Pattern after IGA for CI of mitochondria obtained from frozen rat liver using different digitonin/protein ratios for solubilization. Abbreviations: SC = supercomplex; PAGE = polyacrylamide gel electrophoresis; CI = complex I; IGA = in-gel activity. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66771/66771fig04largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Medium</td>
			<td colspan="3">Composition</td>
		</tr>
		<tr>
			<td>Hypotonic buffer</td>
			<td colspan="3">10 mM MOPS, 83 mM sucrose, pH 7.2</td>
		</tr>
		<tr>
			<td>Hypertonic buffer</td>
			<td colspan="3">30 mM MOPS, 250 mM sucrose, pH 7.2</td>
		</tr>
		<tr>
			<td>Homogenization Buffer A</td>
			<td colspan="3">10 mM Tris, 1 mM EDTA, 0.32 M sucrose, pH 7.4</td>
		</tr>
		<tr>
			<td>Homogenization Buffer AT</td>
			<td colspan="3">225 mM mannitol, 1 mM EGTA, 75 mM sucrose, 0.01% BSA pH 7.4</td>
		</tr>
		<tr>
			<td>Homogenization Buffer AT2</td>
			<td colspan="3">225 mM mannitol, 1 mM EGTA, 75 mM sucrose, 0.02% BSA pH 7.4</td>
		</tr>
		<tr>
			<td>BN sample buffer</td>
			<td colspan="3">50 mM NaCl, 50 mM Imidazole, 2 mM Aminocaproic acid, 1 mM EDTA pH 7.0</td>
		</tr>
		<tr>
			<td>Digitonin solution</td>
			<td colspan="3">10% digitonin in 50 mM NaCl, 50 mM Imidazole, 5 mM Aminocaproic, 4 mM PMSF</td>
		</tr>
		<tr>
			<td>BN loading buffer</td>
			<td colspan="3">5% Coomassie Blue G in 0.75 M aminocaproic acid</td>
		</tr>
		<tr>
			<td>BN cathode buffer A</td>
			<td colspan="3">50 mM tricine, 15 mM Bis-Tris pH =7.0 (4 &#176;C), 0.02% G-250 (Coomassie brilliant blue G-250)</td>
		</tr>
		<tr>
			<td>BN cathode buffer B</td>
			<td colspan="3">50 mM tricine, 15 mM Bis-Tris pH=7.0 (4 &#176;C), 0.002% G-250</td>
		</tr>
		<tr>
			<td>BN anode buffer</td>
			<td colspan="3">50 mM Bis-Tris, pH=7.0</td>
		</tr>
		<tr>
			<td>Complex I IGA substrate</td>
			<td colspan="3">5 mM Tris-HCl pH 7.4, 0.1 mg/mL NADH, 2.5 mg/mL Nitroblue Tetrazolium (NBT)</td>
		</tr>
		<tr>
			<td>Complex II IGA substrate</td>
			<td colspan="3">50 mM Potassium phosphate buffer pH 7.4, 20 mM sodium succinate, 0.2 mM phenazine methosulfate (PMS), 2.5 mg/mL NBT</td>
		</tr>
		<tr>
			<td>Complex III IGA substrate</td>
			<td colspan="3">50 mM Potassium phosphate buffer pH 7.2, 0.05% diaminobenzidine (DAB)</td>
		</tr>
		<tr>
			<td>Complex IV IGA substrate</td>
			<td colspan="3">Same solution as CIII plus 50 &#956;M cytochrome c</td>
		</tr>
		<tr>
			<td>Complex V IGA substrate</td>
			<td colspan="3">35 mM Tris, 270 mM glycine pH 8.3, 14 mM MgSO4, 0.2% Pb(NO3)2, 8 mM ATP&#160;</td>
		</tr>
		<tr>
			<td>Staining solution</td>
			<td colspan="3">Coomassie dye R-250 at 0.25% in 40% methanol and 10% Acetic acid</td>
		</tr>
		<tr>
			<td>Destaining/fixing solution</td>
			<td colspan="3">40% Methanol, 10% Acetic Acid</td>
		</tr>
		<tr>
			<td>Transfer buffer</td>
			<td colspan="3">48 mM Tris, 39 mM Glycine, 20% methanol</td>
		</tr>
	</tbody>
</table>
Table 1: Buffers and media composition.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell type/tissue</td>
			<td>Starting amount</td>
			<td>Expected yield (&#181;g mitochondrial protein)</td>
			<td>Number of lanes (50-60 &#181;g/lane)</td>
		</tr>
		<tr>
			<td>Cultured cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-468</td>
			<td>100 &#181;L vpc</td>
			<td>300-400 &#181;g</td>
			<td>6-9</td>
		</tr>
		<tr>
			<td>143B-derived&#160;</td>
			<td>100 &#181;L vpc</td>
			<td>200-350 &#181;g</td>
			<td>4-7</td>
		</tr>
		<tr>
			<td>Mouse Fibroblasts</td>
			<td>100 &#181;L vpc</td>
			<td>150-200 &#181;g</td>
			<td>3-5</td>
		</tr>
		<tr>
			<td>Heart</td>
			<td>100 mg</td>
			<td>400-500 &#181;g</td>
			<td>10-12*</td>
		</tr>
		<tr>
			<td>Skeletal muscle</td>
			<td>100 mg</td>
			<td>300-400 &#181;g</td>
			<td>8-10*</td>
		</tr>
		<tr>
			<td>Liver</td>
			<td>100 mg</td>
			<td>400-500 &#181;g</td>
			<td>8-10</td>
		</tr>
		<tr>
			<td>Spleen</td>
			<td>50 mg</td>
			<td>150-250 &#181;g</td>
			<td>3-5</td>
		</tr>
		<tr>
			<td>BAT</td>
			<td>100 mg</td>
			<td>200-300 &#181;g</td>
			<td>4-6</td>
		</tr>
		<tr>
			<td>Brain</td>
			<td>100 mg</td>
			<td>250-300 &#181;g</td>
			<td>5-8</td>
		</tr>
	</tbody>
</table>
Table 2:&#160;Expected yields of mitochondria from different cell types and mouse tissues. The mitochondrial fractions obtained from heart or skeletal muscle are purer than those obtained from other tissues. Thus, smaller amounts can be loaded in the gels (*).

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 ...

isolation-mitochondria-for-mitochondrial-supercomplex-analysis-from

Nanyang Technological University

Research

JoVE Journal

Biochemistry

955 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

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most ...

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 ...

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western blotting protocol was optimized and suitable for the analysis of small numbers of HSCs (500 - 15,000 cells). Phenotypic HSCs were purified, accurately counted, and directly lysed in Laemmli sample buffer. Lysates containing equal numbers of cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the blot was prepared and processed following standard Western blotting protocols. Using this protocol, 2,000 - 5,000 HSCs can be routinely analyzed, and in some cases data can be obtained from as few as 500 cells, compared to the 20,000 to 40,000 cells reported in most publications. This protocol should be generally applicable to other hematopoietic cells, and enables the routine analysis of small numbers of cells using standard laboratory procedures.

A standard Western blotting protocol was optimized for analyzing as few as 500 hematopoietic stem or progenitor cells. Optimization involves careful handling of the cell sample, limiting transfers between tubes, and directly lysing the cells in Laemmli sample buffer.

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western ...

Mouse Adipose Tissue Collection and Processing for RNA Analysis

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and Western Blot, since it mostly contains lipids. RNA isolation from adipose tissue samples is also challenging as extra steps are required to avoid these lipids. Here, we present a procedure to collect three anatomically different white adipose tissues from mice, to process these samples and perform RNA isolation. We further describe the synthesis of cDNA and gene expression experiments using real-time PCR. The hereby described protocol allows the reduction of contamination from the animal's hair and blood on fat pads as well as cross-contamination between different fat pads during tissue collection. It has also been optimized to ensure adequate quantity and quality of the RNA extracted. This protocol can be widely applied to any mouse model where adipose tissue samples are required for routine experiments such as real-time PCR but is not intended for isolation from primary adipocytes cell culture.

The purpose of this paper is to present a step-by-step procedure to collect different white adipose tissues from mice, process the fat samples and extract RNA.

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and ...

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 ...

Rapid Isolation of the Mitoribosome from HEK Cells

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondrial genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart.
Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only ~1010 cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.

Mitochondria have specialized ribosomes that diverged from their bacterial and cytoplasmic counterparts. Here we show how mitoribosomes can be obtained from their native compartment in HEK cells. The method involves isolation of mitochondria from suspension cells and consequent purification of mitoribosomes.

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative ...

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 ...

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

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Isolation of Tissue Extracellular Vesicles from the Liver

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