Name: isolation and respiratory measurements of mitochondria from arabidopsis thaliana
Uploaded: 2018-01-05T13:00:00.000+00:00
Duration: 9 min 54 s
Description: Watch this Scientific Journal Video about Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana at JoVE.com

This study was supported by an Australian Research Council Centre of Excellence in Plant Energy Biology CE140100008, an Australian Research Council Future Fellowship (FT130100112) to MWM, and a Feodor Lynen Research Fellowship (Alexander von Humboldt Foundation, Germany) to JS.

This protocol is used for the isolation of intact mitochondria from Arabidopsis thaliana organs grown on soil using continuous colloidal density gradients. All procedures following the collection of the material are carried out at 4 &#176;C.
1. Preparation of Grinding Medium, Wash Buffer, and Gradient Solutions
<ol>
	<li>Prepare 300 mL of grinding medium (minus ascorbate and cysteine) and 200 mL of 2x wash buffer per Table 1 one day prior to the isolation, keep them cold at 4 &#176;C. 
	NOTE: Add sodium ascorbate and L-cysteine (free base) to a final concentration of 17.84 mM and 20.36 mM, respectively, to the grinding medium on the morning of the isolation. If the mitochondria are to be used for western blot or blue native-polyacrylamide gel electrophoresis (BN-PAGE) analyses, make up 2x wash buffer without BSA.</li>
	<li>Prepare gradients on the morning of mitochondrial isolation (Figure 1 and Table 1).
	<ol>
		<li>Make the heavy and light gradient solutions in two beakers; 35 mL of each is sufficient for two gradient tubes.</li>
		<li>Wash the chambers of the gradient pourer and PVC peristaltic tubing with deionized water and ensure even flow through the tubing from all tubing.</li>
		<li>Place 2 centrifuge tubes (50 mL) at a slight angle on ice with the PVC peristaltic tubing outlets taped to the inside of the tubes to ensure the gradient solution runs down the side of the tubes.</li>
		<li>Close the connection between the inner and outer chambers (black lever down). Place gradient pourer on a magnetic stirrer with a small stir bar in the inner chamber.</li>
		<li>Pour the 35 mL heavy gradient solution into the inner chamber (chamber with tubing outlet). Dispense the heavy gradient solution into the two centrifuge tubes placed on ice until half is remaining with the peristaltic pump rate set to fast (300 mL/h).</li>
		<li>Pour the 35 mL light gradient solution into the outer chamber (chamber without tubing outlet). Open the connection between the chambers (push black lever up halfway) and allow solutions to mix gently. Mix the solutions using the magnetic stirrer.</li>
		<li>Allow the solutions to run slowly (60 mL/h) until all of the gradient mix has been dispensed from the chambers resulting in two&#160;0 - 4.4 % (w/v) polyvinylpyrrolidone (PVP) gradient-filled tubes. Keep the gradients on ice until ready to use in Step 2.12. 
		NOTE: Once the gradient has been prepared, dilute 2x wash medium to 1x with prechilled-deionized water and keep cold at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Homogenization and Mitochondrial Isolation
<ol>
	<li>Cut whole rosette tissue from 4-week old Arabidopsis plants grown on soil with scissors, at least 80 plants will be required for 1 prep. 
	NOTE: 10 - 14 day old water cultures, minimum 5 pots/prep, or 2-week old seedlings grown on Murashige &#38; Skoog Basal Salt Mixture (MS) agar plates, minimum of 4 plates, may also be used.</li>
	<li>Place half of the plant material that has been cut into small pieces in a pre-cooled 4 &#176;C large mortar and pestle with 75 mL of grinding medium (Table 1), and grind extensively for several minutes until no big pieces of tissue are left.</li>
	<li>Pre-wet 4 layers of filtration material (22 - 25 &#181;m pore size) with grinding medium and filter homogenate through the 4 layers of filtration material through a plastic funnel into a 500 mL conical flask.</li>
	<li>Grind the remaining half of the tissue with another 75 mL of grinding medium.</li>
	<li>Combine homogenates in the filtration materials and filter as much as possible homogenate through.</li>
	<li>Grind the remaining tissue remaining on the filtration material again with the remaining 150 mL of grinding medium, filter again into the 500 mL conical flask.</li>
	<li>Centrifuge the filtered homogenate in 8 pre-chilled 50 mL plastic centrifuge tubes at 2,500 x g at 4 &#176;C in a fixed angle rotor for 5 min.</li>
	<li>Pour the supernatant into clean centrifuge tubes (50 mL; avoid disturbing the green pellet) and centrifuge at 17,500 x g at 4 &#176;C for 20 min in a fixed angle rotor.</li>
	<li>Discard all but &#60;3 mL of the supernatant by aspiration (or gently pour off without disturbing the pellet). Gently resuspend the pellet in the residual supernatant using a small fine paintbrush.</li>
	<li>Add 10 mL of 1x wash buffer to each tube and combine 4 tubes into 1 clean centrifuge tube (i.e., resulting in 2 tubes).</li>
	<li>Fill these tubes with 1x wash buffer to 50 mL and repeat steps 2.7 - 2.9.</li>
	<li>Pool crude mitochondrial pellets into 1 tube using a dropper and disperse evenly with the fine paintbrush (a small amount of wash buffer may be used but the final volume needs to be less than 3 mL to fit on top of the gradient). Gently layer the mitochondrial suspension with a dropper onto the 2 continuous 0 - 4.4 % (w/v) PVP gradients.</li>
	<li>Balance tubes by weight (using 1x&#160;wash buffer) and centrifuge at 40,000 x g at 4 &#176;C for 40 min. Ensure the break is turned off. Mitochondria will migrate to a band near the bottom of the tube, or may be present in the heavy solution at the bottom of the tube (identified by a cloudy, yellowish band; Figure 2).</li>
	<li>Carefully remove the upper 5 cm of solution above the mitochondrial band by aspiration.</li>
	<li>Distribute the remaining solution containing the mitochondria into 2 clean tubes and fill with 1x wash buffer. Evenly distribute the colloidal density gradient and mitochondria by covering the mouth of the tube with a plastic paraffin film and inverting several times. Centrifuge for 15 min at 31,000 x g at 4 &#176;C with slow braking. 
	NOTE: Before centrifugation, make sure the mitochondria and the remaining colloidal density gradient are evenly distributed. Otherwise, the colloidal density gradient may concentrate at the bottom of the tube and prevent pelleting of the mitochondria.</li>
	<li>Remove the supernatant by aspiration, fill the tube with 1x wash buffer up to 50 mL, and invert again to ensure mixing. Centrifuge for 15 min at 31,000 x g at 4 &#176;C with slow braking.</li>
	<li>Aspirate the supernatant and collect the mitochondrial pellets in as small a volume (~500 &#181;L) as possible using a pipette with modified 200 &#181;L tips (cut the tip a little above its end to increase its opening). Place the mitochondria into a 1.5 mL tube and keep on ice for immediate use or store at -80 &#176;C. 
	NOTE: If mitochondria will be used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), BN-PAGE, or immunoblot analyses, use 1x wash buffer without BSA for the final washes (i.e., step 2.15 and 2.16).</li>
	<li>Determine the protein concentration of mitochondria using the Bradford method. 
	NOTE: For BN-PAGE centrifuge aliquots at 4 &#176;C, and store pellet at -80 &#176;C until use. For oxygen consumption measurements, only freshly isolated mitochondria can be used.</li>
</ol>
3. Oxygen Consumption Measurements
NOTE: Oxygen consumption by freshly isolated plant mitochondria can be analyzed with a Clark-type oxygen electrode, enabling the determination of mitochondrial intactness, cytochrome c pathway activity, and alternative pathway activity.
<ol>
	<li>Software installation
	<ol>
		<li>Install licensed oxygen monitoring software onto the computer used for oxygen consumption measurements.</li>
	</ol>
	</li>
	<li>Preparation of respiration medium, substrates, chemical stock solutions, and the Clark-type oxygen electrode
	<ol>
		<li>Prepare respiration medium (300 mM sucrose, 10 mM NaCl, 5 mM KH2PO4, 2 mM MgSO4, 0.1 % (w/v) bovine serum albumin (BSA), 10 mM TES (pH 7.2)) used for oxygen consumption assays.</li>
		<li>Prepare substrates, inhibitors, and effectors used for measuring mitochondrial respiration (Table 2). These can be prepared earlier and stored at -20 &#176;C. 
		NOTE: Neutralize the pH of acidic solutions such as organic acids to pH 7.0 using NaOH before use.</li>
		<li>Warm the respiration medium to the same temperature as the assay chamber (25 &#176;C).</li>
		<li>Assemble the Clark-type oxygen electrode as described in the protocol of the manufacturer.</li>
		<li>Place 1 mL air-saturated water (air-saturated water is obtained by vigorously shaking a small quantity of deionized water in a large conical flask) in the measuring chamber. To switch on the stirrer, click the &#34;Stirrer Speed&#34; button. Click the &#34;On&#34; button and set the stirrer speed to 65 rpm. Click &#34;OK&#34; to continue. Stir the water continuously at 25 &#176;C.</li>
	</ol>
	</li>
	<li>Calibration of oxygen electrode
	<ol>
		<li>Warm the water that will be used for calibration to the same temperature as the assay chamber (25 &#176;C).</li>
		<li>Wait for the current to stabilize and calibrate the oxygen electrode.</li>
		<li>To start calibration of the oxygen electrode, click the &#34;Calibrate&#34; button, select &#34;Liquid Phase Calibration,&#34; and then &#34;Air Saturated Water.&#34;</li>
		<li>A new window will open (Oxygen Calibration (Liquid Phase) - Step 1 of 5). Select the channel to calibrate (the channel the electrode control box is attached to) and enter variables (set &#34;Chamber Temperature&#34; to 25 &#176;C and &#34;Atmospheric Pressure&#34; to 101.32 kPa). Click &#34;OK &#34;to continue.</li>
		<li>In the next step (Step 2 of 5), setup the stirrer speed (65 rpm) and click &#34;OK&#34; to continue.</li>
		<li>In step 3 of 5, wait for the signal to reach a plateau. The term &#34;Plateau reached, press OK to continue&#34; will appear in the box. Click &#34;OK&#34; to continue.</li>
		<li>In step 4 of 5, establish zero oxygen in the chamber by adding 5 mg sodium dithionite. Click &#34;OK&#34; to continue. 
		NOTE: A decline in oxygen concentration should be visible in the trace and should reach 0.</li>
		<li>In the last step of calibration (Step 5 of 5), wait for the signal to reach a plateau. The term &#34;Plateau reached, press OK to continue&#34; will appear in the box. Click &#34;OK&#34; to continue.</li>
		<li>Click &#34;Save Calibration&#34; to save the calibration. A new window will open. Click &#34;OK&#34; to confirm to save the new calibration for the oxygen control box. 
		NOTE: After successful calibration, the labeling &#34;Oxygen (nmol/mL)&#34; will appear on the y-axis.</li>
		<li>After calibration, clean the measuring chamber by rinsing the chamber with water (at least 5 - 10 times) to ensure proper cleaning. Place 1 mL of respiration medium into the measuring chamber and allow the membrane to equilibrate for a few minutes until the oxygen consumption trace is linear.</li>
		<li>Adapt the scale of the oxygen consumption trace to get a good slope view. Click &#34;Zoom XY&#34; and enter variables (set &#34;Oxygen&#34; to 0 - 300 and the &#34;Time Axis&#34; to 0 - 45 min).</li>
	</ol>
	</li>
	<li>Determination of mitochondrial integrity
	<ol>
		<li>With plunger open, add 970 &#181;L air-saturated respiration medium to the reaction chamber.</li>
		<li>Add 30 &#181;L isolated mitochondria or 150 &#181;g of total mitochondrial protein as determined after mitochondria isolation (total protein needs to be included in calculations afterwards) to the reaction chamber using a pipette with a cut tip.</li>
		<li>Stir the mitochondrial suspension continuously using a magnetic stirrer bar (65 rpm) at 25 &#176;C to air-saturate the solution.</li>
		<li>Seal the chamber with the plunger, click &#34;Start Recording&#34; to record oxygen consumption, and allow the oxygen consumption trace to stabilize (1 - 2 min).</li>
		<li>Determine the rate of oxygen consumption manually after adding 10 mM ascorbate and 25 &#181;M cytochrome c for 5 min to get the &#34;before detergent&#34; rate. 
		NOTE: To label the addition of substrates, inhibitors, and effectors directly in the trace, click the &#34;Add Event Mark&#34; button and enter the corresponding label designation. Click &#34;OK&#34; to continue.</li>
		<li>Determine the rate of oxygen consumption manually for 3 min after adding 0.05% (v/v) detergent to give the &#34;after detergent&#34; rate. Click &#34;Stop Recording.&#34;</li>
		<li>Click the &#34;Rates of Change Table&#34; button. 2 cursors will appear as vertical lines on the graph screen. Click and drag the pair of rate cursors to the required positions in the trace to define the rate interval. Move the rate interval along the trace using the left rate cursor. Set the rate interval itself using the right rate cursor. The oxygen monitoring software automatically draws a line of best fit between the two cursors while moving the rate interval cursors along the trace.</li>
		<li>Once the desired rate interval and position has been defined, enter the rate into the table with a right click on 1 of the 2 cursors, and select &#34;Add Rate to Table.&#34;&#160;Click the &#34;Add&#34; button to confirm to display the rate on the main rate table.</li>
		<li>Calculate mitochondrial integrity by dividing the &#34;before detergent&#34; rate by the &#34;after detergent&#34; rate, expressed as a percentage, and subtract it from 100.</li>
	</ol>
	</li>
	<li>Determination of mitochondrial respiration
	<ol>
		<li>With plunger open, add 970 &#181;L of air-saturated respiration medium to the reaction chamber. Add 500 &#181;M ATP and 30 &#181;L isolated mitochondria or 150 &#181;g of total mitochondrial protein using a pipette with a cut tip to the respiration medium in the reaction chamber.</li>
		<li>Stir the mitochondrial suspension continuously using a magnetic stirrer bar (65 rpm) at 25 &#176;C.</li>
		<li>Close plunger, click &#34;Start Recording&#34; to record oxygen consumption, and wait for 1 - 2 min for oxygen consumption rate to stabilize (when the oxygen consumption trace is linear). Add 5 mM succinate. Record the rate of oxygen consumption for about 2 min. 
		NOTE: To label the addition of substrates, inhibitors, and effectors directly in the trace, click the &#34;Add Event Mark&#34; button and enter the corresponding label designation. Click &#34;OK&#34; to continue.</li>
		<li>Add 1 mM adenosine diphosphate (ADP). Continue recording the rate of oxygen consumption for 2 min.</li>
		<li>Add 1 mM NADH. Record the rate of oxygen consumption for 4 - 8 min. This rate is the capacity of respiration via cytochrome oxidase.</li>
		<li>Add 1 mM potassium cyanide (KCN) (or 2.5 &#181;M myxothiazol or 5 &#181;M antimycin A) to inhibit the cytochrome c pathway, and continue to record for about 2 min to observe oxygen consumption via the alternative oxidase.</li>
		<li>Add 5 mM dithiothreitol (DTT) and 10 mM pyruvate to fully activate the alternative oxidase. Continue to record for 5 - 7 min: This is the capacity of the alternative oxidase.</li>
		<li>Add 500 &#181;M n-propyl gallate (nPG), and record for 2 min. This assesses residual oxygen consumption rate that cannot be chemically inhibited. Subtract this rate from the other rates recorded, as it is not likely to be mitochondrial in origin. Oxygen consumption still occurring after inhibition of the cytochrome c and AOX pathway (by KCN and nPG) is very likely to come from peroxidases present as contaminants in the isolated mitochondria9.</li>
		<li>Click &#34;Stop Recording.&#34;</li>
		<li>Click the &#34;Rates of Change Table&#34; button and enter the rate into the table with a right click on one of the two cursors, and select &#34;Add Rate to Table&#34;. Click the &#34;Add&#34; button to confirm to display the rate on the main rate table. 
		NOTE: When adding effectors dissolved in organic solvents (e.g., antimycin A, nPG, myxothiazol), the chamber and plunger must be rinsed with ethanol and then water to ensure proper cleaning.</li>
	</ol>
	</li>
</ol>

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The authors declare no conflict of interests.

Typically, isolation of mitochondria from Arabidopsis leaves yields up 3 mg of mitochondria from approximately 80 - 100 3 - 4-week old plants, although yields of greater than 5 mg can often be achieved with thorough grinding. The yield varies with growth conditions and decreases dramatically as leaves senesce, although mitochondria structure seems to be well maintained during senescence9. One of the most critical features to obtain a good yield is the method of grinding to lyse cells to release mi...

The history of plant mitochondrial research goes back over 100 years1. Intact mitochondria were first isolated in the early 1950s using differential centrifugation. The advent of a colloidal density gradient in the 1980s allowed mitochondria to be purified without suffering osmotic adjustment. While gradient purified mitochondria are suitable for most purposes, due to the sensitivity of mass spectrometry, even relatively minor contaminants can be detected and may be inappropriately assigned a mitochondrial location2. The use of free flow electrophoresis can remove both plastidic and peroxisome contamination3, but free flow electrophoresis is a highly specialized technique and is not required for the vast majority of studies. Furthermore, when determining the location of a protein it needs to be remembered that dual or multiple targeting of proteins occurs in cells. Over 100 dual targeted proteins are described for chloroplasts/plastids and mitochondria4, and a number of proteins targeted to mitochondria and peroxisomes are also known5. Furthermore, the re-location of proteins under specific stimuli, e.g. oxidative stress, is an emerging theme in cell biology6. Thus, the location of proteins needs to be considered in the context of the biology studied, and a variety of approaches are used to determine and verify location2.
Mitochondria are typically isolated from plant tissues by homogenization, a balance is required between breaking open the cell wall to release mitochondria, and not damaging the mitochondria. Traditionally, with potato and cauliflower, homogenization involves using household blender/juicer apparatus to make a liquid extract in a buffer with various components to maintain activity.&#160;Isolation of mitochondria from pea leaves, (a popular material for mitochondrial isolation using young seedlings (~10 days old), utilizes a blender to lyse cells as the leaf material is soft. With the availability of Arabidopsis thaliana T-DNA insertional knock-out lines, the need to be able to purify mitochondria to carry out functional studies has necessitated the development of methods to isolate mitochondria from leaf, root or flower tissue. Overall the methods developed for other plants worked well7, with the perquisite that grinding of the material needed to be optimized. For Arabidopsis this can be achieved in a variety of ways (see below), and differs between tissue types (root versus shoot). The use of the continuous gradient can also be optimized as the density of mitochondria from different organs or developmental stages means they can migrate differently. Thus, for maximum separation the density of the gradient can be refined to ensure to achieve best separation.
Once purified the mitochondria can be used for a variety of studies, including protein and tRNA uptake experiments, enzyme activity assays, respiratory chain measurements and western blot analyses. Isolated mitochondria can also be used for mass spectrometry analyses of protein abundance. Targeted multiple reaction monitoring (MRM) analyses allows for the quantification of defined proteins, but require significant assay development. In contrast, quantification by dimethyl or other isotope labels8, provides a discovery approach in identifying differences across the whole proteome that can be used to uncover novel biological insights.

<table><tbody><tr><td>ADP</td><td>Sigma-Aldrich</td><td>A2754</td><td>Chemical</td></tr><tr><td>Antimycin A</td><td>Sigma-Aldrich</td><td>A8674</td><td>Chemical, dissolve in ethanol</td></tr><tr><td>AOX antibody</td><td>from Tom Elthon</td><td></td><td>Elthon et al., 1989</td></tr><tr><td>Ascorbate</td><td>Sigma-Aldrich</td><td>A0157</td><td>Ascorbate Oxidase from Cucurbita sp.</td></tr><tr><td>ATP</td><td>Sigma-Aldrich</td><td>A26209</td><td>Chemical</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Bovogen</td><td>BSAS 1.0</td><td>Chemical</td></tr><tr><td>Clarity western ECL substrate</td><td>Bio-Rad Laboratories</td><td>1705061</td><td>Chemical</td></tr><tr><td>Criterion Stain-Free Precast Gels 8-16% 18 Wells</td><td>Bio-Rad Laboratories</td><td>5678104</td><td>Chemical</td></tr><tr><td>Cyanide</td><td>Sigma-Aldrich</td><td>60178</td><td>Chemical</td></tr><tr><td>Cytochrome c</td><td>Sigma-Aldrich</td><td>C3131</td><td>Chemical</td></tr><tr><td>Difco Agar, granulated</td><td>BD Biosciences</td><td>214530</td><td>Chemical</td></tr><tr><td>Dithiotreitol</td><td>Sigma-Aldrich</td><td>D0632</td><td>Chemical</td></tr><tr><td>EDTA disodium salt</td><td>Sigma-Aldrich</td><td>E5134</td><td>Chemical</td></tr><tr><td>Gamborg B-5 Basal Medium</td><td>Austratec</td><td>G398-100L</td><td>Chemical</td></tr><tr><td>Gamborg Vitamin Solution (1000x)</td><td>Austratec</td><td>G219-100ML</td><td>Chemical</td></tr><tr><td>Goat Anti-Mouse IgG (H + L)-HRP Conjugate</td><td>Bio-Rad Laboratories</td><td>1706516-2ml</td><td>Chemical</td></tr><tr><td>Goat Anti-Rabbit IgG (H + L)-HRP Conjugate</td><td>Bio-Rad Laboratories</td><td>1706515-2ml</td><td>Chemical</td></tr><tr><td>L-Cysteine</td><td>Sigma</td><td>C7352-100G</td><td>Chemical</td></tr><tr><td>Magnesium sulfate</td><td>Sigma-Aldrich</td><td>230391</td><td>Chemical</td></tr><tr><td>Murashige &amp;amp; Skoog Basal Salt Mixture (MS)</td><td>Austratec</td><td>M524-100L</td><td>Chemical</td></tr><tr><td>Myxothiazol</td><td>Sigma-Aldrich</td><td>T5580</td><td>Chemical, dissolve in ethanol</td></tr><tr><td>NADH</td><td>Sigma-Aldrich</td><td>N8129</td><td>Chemical</td></tr><tr><td>Ndufs4 antibody</td><td>from Etienne Meyer</td><td></td><td>Meyer et al., 2009</td></tr><tr><td>n-Propyl gallate</td><td>Sigma-Aldrich</td><td>P3130</td><td>Chemical, dissolve in ethanol</td></tr><tr><td>Percoll</td><td>GE Healthcare</td><td>17-0891-01</td><td>Chemical, colloidal density gradient</td></tr><tr><td>Polyvinylpyrrolidone (PVP40)</td><td>Sigma-Aldrich</td><td>PVP40</td><td>Chemical</td></tr><tr><td>Potassium cyanide</td><td>Sigma-Aldrich</td><td>60178</td><td>Chemical</td></tr><tr><td>Potassium phosphate monobasic (KH2PO4)</td><td>Sigma-Aldrich</td><td>P5655</td><td>Chemical</td></tr><tr><td>Pyruvate</td><td>Sigma-Aldrich</td><td>P2256</td><td>Chemical</td></tr><tr><td>Sodium chloride</td><td>Chem-Supply</td><td>SA046</td><td>Chemical</td></tr><tr><td>Sodium dithionite</td><td>Sigma-Aldrich</td><td>157953</td><td>Chemical</td></tr><tr><td>Sodium L-ascorbate</td><td>Sigma</td><td>A4034-100G</td><td>Chemical</td></tr><tr><td>Succinate</td><td>Sigma-Aldrich</td><td>S2378</td><td>Chemical</td></tr><tr><td>Sucrose</td><td>Chem-Supply</td><td>SA030</td><td>Chemical</td></tr><tr><td>TES</td><td>Sigma-Aldrich</td><td>T1375</td><td>Chemical</td></tr><tr><td>Tetrasodium pyrophosphate (Na4P2O7 &amp;middot; 10H2O)</td><td>Sigma-Aldrich</td><td>221368</td><td>Chemical</td></tr><tr><td>Trans-Blot Turbo RTA Midi Nitrocellulose Transfer Kit</td><td>Bio-Rad Laboratories</td><td>1704271</td><td>Chemical</td></tr><tr><td>Triton-X 100</td><td>Sigma-Aldrich</td><td>X100</td><td>Chemical, detergent</td></tr><tr><td>Western Blocking Reagent</td><td>Sigma</td><td>11921681001</td><td>Chemical</td></tr><tr><td>Balance</td><td>Mettler Toledo</td><td>XS204</td><td>Equipment</td></tr><tr><td>Beakers</td><td>Isolab</td><td>50 mL</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman Coulter</td><td>Avanti J-26XP</td><td>Equipment</td></tr><tr><td>Centrifuge tubes</td><td>Nalgene</td><td>3117-9500</td><td>Equipment</td></tr><tr><td>Circulator</td><td>Julabo</td><td>1124971</td><td>Attached to oxygen electrode chamber</td></tr><tr><td>Conical flask</td><td>Isolab</td><td>500 mL</td><td></td></tr><tr><td>Dropper</td><td></td><td>3 mL</td><td></td></tr><tr><td>Fixed angle rotor</td><td>Beckman Coulter</td><td>JA25.5</td><td>Equipment</td></tr><tr><td>Funnel</td><td>Per Alimenti</td><td>14 cm</td><td>For filtering</td></tr><tr><td>Gradient pourer</td><td>Bio-Rad</td><td>165-4120</td><td>For preparation of gradients</td></tr><tr><td>Magnetic Stirrer ATE</td><td>VELP Scientifica</td><td>F20300165</td><td>Equipment</td></tr><tr><td>Miracloth</td><td>VWR</td><td>EM475855-1R</td><td>Filtration material</td></tr><tr><td>Mortar and pestle</td><td>Jamie Oliver</td><td>Granite, 6 Inch</td><td>Equipment</td></tr><tr><td>O2view</td><td>Hansatech Instruments</td><td></td><td>Oxygen monitoring software</td></tr><tr><td>Oxygraph Plus System</td><td>Hansatech Instruments</td><td>1187253</td><td>Clark-type oxygen electrode</td></tr><tr><td>Paintbrush</td><td>Artist first choice</td><td>1008R-12</td><td></td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM-996</td><td>plastic paraffin film</td></tr><tr><td>Peristaltic pump</td><td>Gilson</td><td>F155001</td><td>For preparation of gradients</td></tr><tr><td>PVC peristaltic tubing</td><td>Gilson</td><td>F117930</td><td>For preparation of gradients</td></tr><tr><td>Water bath</td><td>VELP Scientifica</td><td>OCB</td><td>Equipment</td></tr></tbody></table>

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.

Using this protocol, we were able to detect different mitochondrial proteins by SDS-PAGE and immunoblotting. As shown in Figure 3A, the protein isolated from water culture tissue is sufficient to detect a faint band (2 &#181;g). Signal intensity increases proportionately to the amounts loaded. For mitochondria isolated from tissues grown on plates (Figure 3B), the response to high light stress treatment in different genotypes can...

Watch this Scientific Journal Video about Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana at JoVE.com

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.

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 ...

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Cell Biology

Biochemistry

Unit 1

Mitochondria and Energy Production

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15.2K Views.  La Trobe University. Mitochondriën zijn slechts een klein percentage van de plant cel, moeten ze voor een aantal studies worden gezuiverd. Mitochondriën kunnen worden geïsoleerd uit een verscheidenheid van organen van de plant door homogenisering, gevolgd door de differentiaal- en dichtheid kleurovergang centrifugeren te verkrijgen van een hoogst gezuiverde mitochondriale fractie.

Video: Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Developmental Biology

Mitochondrial Isolation from Skeletal Muscle

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

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

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

Biology

Mitochondrial Fractionation in Small Tissue Samples for Supercomplex Analysis

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

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

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

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

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

Mitochondrial Isolation from Rodents for High-Quality Bioenergetic Studies

Robust Mitochondrial Isolation from Rodent Cardiac Tissue

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

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

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

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

Changing Mitochondrial Function for Therapeutic and Bioinsecticide Applications

Unveiling Xenobiotic Transport and Effects in Isolated Mitochondria: Insights from Respirometric and Enzymatic Assays

Mitochondria are often referred to as the cell's powerhouse due to their crucial role in energy production through the electron transport chain (ETC). However, their significance extends far beyond energy production. Dysregulation of mitochondrial bioenergetics can trigger intracellular cascades that impact health and cellular function. Given their physiological importance, there is growing interest in exploring the pharmacological potential of mitochondrial function for developing new therapies and bioproducts. Modulating mitochondrial bioenergetics could provide innovative approaches to address challenges such as neurodegenerative disorders, metabolic diseases, and cancer, as well as to develop bioproducts for controlling pests and vector-borne diseases.
This work presents a method for assessing the effects of xenobiotics on the electron transport chain (ETC) using various substrates, including glutamate and NADH for complex I, succinate for complex II, and cytochrome c (both oxidized and reduced) for complexes III and IV. This methodology allows the activation or inhibition of electron transport through mitochondrial complexes to be evaluated using a respirometer and spectrophotometer. The mitochondria can be sourced from isolated mitochondria, fragmented cells, or homogenized tissue from various species.
In the laboratory, mitochondrial function has been analyzed in Aedes aegypti and Wistar rats, but this method is also applicable to other species, such as Rhipicephalus microplus and Rhodnius prolixus. This approach provides the basis for theorizing about the existence of uncoupler proteins and species-specific oxidizable substrate preferences influenced by their unique energetic demands. Recent findings offer valuable insights into innovative bioinsecticide design strategies that target mitochondrial function, holding significant potential for effectively controlling vector-borne diseases and pest infestations.

This study introduces a methodology for studying xenobiotic effects on mitochondrial function via respirometric and enzymatic assays. It examines electron transport chain modulation in various species, offering insights for targeted biopesticide development against insect vector-borne diseases.

Mitochondria are often referred to as the cell's powerhouse due to their crucial role in energy production through the electron transport chain (ETC). ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized near the mitochondria. Support for this possibility is derived from recent studies, in which many mRNAs encoding mitochondrial proteins were shown to be localized to the mitochondria vicinity. Together with earlier demonstrations of ribosomes&#8217; association with the outer membrane, these results suggest a localized translation process. Such localized translation may improve import efficiency, provide unique regulation sites and minimize cases of ectopic expression. Diverse methods have been used to characterize the factors and elements that mediate localized translation. Standard among these is subcellular fractionation by differential centrifugation. This protocol has the advantage of isolation of mRNAs, ribosomes and proteins in a single procedure. These can then be characterized by various molecular and biochemical methods. Furthermore, transcriptomics and proteomics methods can be applied to the resulting material, thereby allow genome-wide insights. The utilization of yeast as a model organism for such studies has the advantages of speed, costs and simplicity. Furthermore, the advanced genetic tools and available deletion strains facilitate verification of candidate factors.

Many mRNAs encoding mitochondrial proteins are associated with the mitochondria outer membrane. We describe a subcellular fractionation procedure aimed at isolation of yeast mitochondria with its associated mRNAs and ribosomes. This protocol can be applied to cells grown under diverse conditions&#160;in order to reveal mechanisms of mRNA localization and localized translation near the mitochondria.

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized ...

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

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

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

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

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

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

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

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

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

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

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

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

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