mitochondrial isolation: a technique to isolate mitochondria from human ovarian cancer tissue sample

Mitochondrial Isolation: A Technique to Isolate Mitochondria from Human Ovarian Cancer Tissue Sample

To begin this procedure, prepare 250 milliliters of the mitochondrial isolation buffer as outlined in the text protocol. Place approximately 1.5 grams of ovarian cancer tissues in a clean glass dish. Add 2 milliliters of pre-chilled mitochondrial isolation buffer to lightly wash the blood from the tissue surface. Repeat this wash three times.
Then, use clean ophthalmic scissors to fully mince the tissue into pieces that are approximately one cubic millimeter and transfer the minced tissues into a 50-milliliter centrifuge tube. Add 13.5 milliliters of mitochondrial isolation buffer containing nagarse at a concentration of 0.2 milligrams per milliliter. Use an electric homogenizer to homogenize the minced tissues for two minutes at 4 degrees Celsius.
After this, add another 3 milliliters of mitochondrial isolation buffer to the tissue homogenates and mix them well by pipetting. Centrifuge the prepared tissue homogenate at 1,300 x g and at 4 degrees Celsius for 10 minutes. Remove the pellet and keep the supernatant.
Next, centrifuge the supernatant at 10,000 x g and at 4 degrees Celsius for 10 minutes. Remove the microsome-containing supernatant and keep the pellet. Add 2 milliliters of mitochondrial isolation buffer and resuspend the pellet thoroughly by light pipetting.
Centrifuge this pellet suspension at 7,000 x g and at 4 degrees Celsius for 10 minutes. Discard the supernatant and keep the pellet, which contains the crude mitochondria. Add 12 milliliters of 25% density gradient medium to resuspend the extracted crude mitochondria. Make a discontinuous density gradient containing the resuspended crude mitochondria, as outlined in the text protocol, and centrifuge it at 52,000 x g and at 4 degrees Celsius for 90 minutes.
Use a long and blunt syringe to collect the purified mitochondria at the interface between the 25% and 30% gradient mediums and transfer this to a clean tube. Add mitochondrial isolation buffer to the collected mitochondria to dilute it to a three-fold volume. Centrifuge at 15,000 x g and at 4 degrees Celsius for 20 minutes. Discard the supernatant and keep the pellet. Collect the final pellet, which contains the purified mitochondria, and store it at minus 20 degrees Celsius.

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All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Preparation of mitochondria from human ovarian cancer tissues
Ovarian tissue samples including ovarian cancer tissues (n = 7) were used for this protocol.
<ol>
	<li>Prepare 250 mL of the mitochondrial isolation buffer by mixing 210 mM mannitol, 70 mM sucrose, 100 mM potassium chloride (KCl), 50 mM Tris-HCl, 1 mM diamine tetraacetic acid (EDTA), 0.1 mM ethylene glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA), 1 mM phenylmethanesulfonyl fluoride (PMSF) protease inhibitor, 2 mM sodium orthovanadate (V), and 0.2% bovine serum albumin (BSA), pH 7.4.</li>
	<li>Place ~1.5 g of ovarian cancer tissues in a clean glass dish.</li>
	<li>Add 2 mL of the pre-chilled mitochondrial isolation buffer to lightly wash the blood from the tissue surface 3x.</li>
	<li>Use clean ophthalmic scissors to fully mince the tissue into about 1 mm3 pieces, and transfer the minced tissues into a 50 mL centrifuge tube.</li>
	<li>Add 13.5 mL of mitochondrial isolation buffer containing 0.2 mg/mL nagarse, and then use an electric homogenizer to homogenize (use scale 2, 10 s 6x, interval 10 s) the minced tissues (2 min, 4 &#176;C).</li>
	<li>Add another 3 mL of mitochondrial isolation buffer into the tissue homogenates and mix them well by pipetting.</li>
	<li>Centrifuge the prepared tissue homogenate (1,300 x g, 10 min, 4 &#176;C). Remove the crude nuclear fraction (i.e., the pellet) and keep the supernatant.</li>
	<li>Recentrifuge the supernatant (10,000 x g, 10 min, 4 &#176;C). Remove the microsomes (i.e., the supernatant) and keep the pellet.</li>
	<li>Add 2 mL of mitochondrial isolation buffer and resuspend the pellet well by light pipetting.</li>
	<li>Centrifuge the pellet suspension (7,000 x g, 10 min, 4 &#176;C). Discard the supernatant and keep the crude mitochondria (i.e., the pellet).</li>
	<li>Add 12 mL of 25% density gradient medium (i.e., Nycodenz) to resuspend the extracted crude mitochondria.</li>
	<li>Make a discontinuous density gradient by filling a tube from bottom to top with 5 mL of 34%, 8 mL of 30%, 12 mL of 25% (containing the crude mitochondria from step 1.11), 8 mL of 23%, and 3 mL of 20% density gradient medium, and centrifuge it (52,000 x g, 90 min, 4 &#176;C).</li>
	<li>Use a long and blunt syringe to collect the purified mitochondria at the interface between the 25% and 30% density gradient medium into a clean tube.</li>
	<li>Add mitochondrial isolation buffer into the collected mitochondria to dilute it to a three-fold volume. Centrifuge (15,000 x g, 20 min, 4 &#176;C) and discard the supernatant.</li>
	<li>Add 2 mL of mitochondrial isolation buffer to resuspend the pellet and centrifuge (15,000 x g, 20 min, 4 &#176;C). Discard the supernatant and keep the pellet.</li>
	<li>Collect the final pellet (i.e., the purified mitochondria) and store it at -20 &#176;C. 
	NOTE: Combine all purified mitochondria from the ovarian cancer tissue as the mitochondria sample for quantitative proteomics analysis.</li>
</ol>

<table><tbody><tr><td>Diamine tetraacetic acid (EDTA)</td><td>Sigma</td><td>798681-100G</td><td>Anhydrous, free-flowing, Redi-Dri, &amp;ge;98%</td></tr><tr><td>DTT</td><td>Sigma</td><td>10197777001</td><td>1,4-Dithiothreito</td></tr><tr><td>Ethylen glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA)</td><td>Sigma</td><td>E0396-10G</td><td>BioXtra, &amp;ge;97 .0%</td></tr><tr><td>Homogenizer</td><td>SilentShake</td><td>HYQ-3110</td><td></td></tr><tr><td>Low-temperature super-speed centrifuge</td><td>Eppendorf</td><td>5424R</td><td></td></tr><tr><td>Centrifuge</td><td>XiangYi</td><td>TDZ4--WS</td><td></td></tr><tr><td>Mannitol</td><td>Macklin</td><td>M813424-100G</td><td>Mannitol is a polyol (polyhydric alcohol) produced from hydrogenation from fructose that functions as a sweetener, humectant, and bulking agent. It has low hygroscopicity and poor oil solvency.</td></tr><tr><td>Phenylmethanesulfonyl fluoride (PMSF) protease inhibitor</td><td>Solarbio</td><td>P0100-1ML</td><td>PMSF is a protease inhibitor that reacts with serine residues to inhibit trypsin, chymotrypsin, thrombin, and papain.</td></tr><tr><td>Potassium chloride</td><td>Macklin</td><td>P816354-25G</td><td>Potassium chloride, KCI, also known as potassium muriate and sylvite, is a colorless crystalline solid with a salty taste that melts at 776&amp;deg;C (1420 OF). It is soluble in water, but insoluble in alcohol. Potassium chloride is used in fertilizers, pharmaceuticals, photography, and as a salt substitute</td></tr></tbody></table>

Source: Zhan, X. et al. <a href="https://www.jove.com/t/60435">Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis.</a> J. Vis. Exp. (2019)
This video describes the procedure for isolating mitochondria from human ovarian cancer tissues using differential-speed centrifugation in combination with density gradient centrifugation. The isolated mitochondria can be used for proteomic analysis of human ovarian cancer mitochondrial proteome.

Source: Zhan, X. et al.  Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis. J. Vis. ...

Mitochondria, the cytoplasmic organelles known as the cell&#39;s powerhouses, undergo drastic structural and functional changes during cancer progression.
To isolate mitochondria, take clean cancerous tissue in a glass dish and mince it into small pieces. Transfer the pieces into a tube containing mitochondrial isolation buffer. Homogenize the sample to disrupt the cells and release their contents, including the organelles.
Centrifuge the mixture at a low speed. This pelletizes the denser components like nuclei, tissue debris, and any intact cells, leaving less dense components like mitochondria, microsomes, peroxisomes, and lysosomes in the supernatant. Collect the supernatant and centrifuge at high speed. Microsomes, being relatively lighter, remain in the supernatant, while the crude mitochondria-containing fraction pelletizes at the bottom.
Remove the supernatant and resuspend the pellet in a suitable density gradient medium. Set up a discontinuous density gradient by layering the crude mitochondrial fraction within different density media, arranging them in decreasing order of densities from bottom to top. Centrifuge at a very high speed. Organelles separate within the tube to adopt an equilibrium position based on their relative densities. Collect the mitochondrial fraction formed at the interface between the density media, containing peroxisomes and lysosomes.

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Mitochondrial Isolation: A Technique to Isolate Mitochondria from Human Ovarian Cancer Tissue Sample

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Mitochondrial Isolation: A Technique to Isolate Mitochondria from Human Ovarian Cancer Tissue Sample