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

Overview

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.

Protocol

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.

1. 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.
2. Place ~1.5 g of ovarian cancer tissues in a clean glass dish.
3. Add 2 mL of the pre-chilled mitochondrial isolation buffer to lightly wash the blood from the tissue surface 3x.
4. 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.
5. 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 °C).
6. Add another 3 mL of mitochondrial isolation buffer into the tissue homogenates and mix them well by pipetting.
7. Centrifuge the prepared tissue homogenate (1,300 x g, 10 min, 4 °C). Remove the crude nuclear fraction (i.e., the pellet) and keep the supernatant.
8. Recentrifuge the supernatant (10,000 x g, 10 min, 4 °C). Remove the microsomes (i.e., the supernatant) and keep the pellet.
9. Add 2 mL of mitochondrial isolation buffer and resuspend the pellet well by light pipetting.
10. Centrifuge the pellet suspension (7,000 x g, 10 min, 4 °C). Discard the supernatant and keep the crude mitochondria (i.e., the pellet).
11. Add 12 mL of 25% density gradient medium (i.e., Nycodenz) to resuspend the extracted crude mitochondria.
12. 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 °C).
13. 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.
14. Add mitochondrial isolation buffer into the collected mitochondria to dilute it to a three-fold volume. Centrifuge (15,000 x g, 20 min, 4 °C) and discard the supernatant.
15. Add 2 mL of mitochondrial isolation buffer to resuspend the pellet and centrifuge (15,000 x g, 20 min, 4 °C). Discard the supernatant and keep the pellet.
16. Collect the final pellet (i.e., the purified mitochondria) and store it at -20 °C.
    NOTE: Combine all purified mitochondria from the ovarian cancer tissue as the mitochondria sample for quantitative proteomics analysis.

Materials

Name	Company	Catalog Number	Comments
Diamine tetraacetic acid (EDTA)	Sigma	798681-100G	Anhydrous, free-flowing, Redi-Dri, ≥98%
DTT	Sigma	10197777001	1,4-Dithiothreito
Ethylen glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA)	Sigma	E0396-10G	BioXtra, ≥97 .0%
Homogenizer	SilentShake	HYQ-3110
Low-temperature super-speed centrifuge	Eppendorf	5424R
Centrifuge	XiangYi	TDZ4--WS
Mannitol	Macklin	M813424-100G	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.
Phenylmethanesulfonyl fluoride (PMSF) protease inhibitor	Solarbio	P0100-1ML	PMSF is a protease inhibitor that reacts with serine residues to inhibit trypsin, chymotrypsin, thrombin, and papain.
Potassium chloride	Macklin	P816354-25G	Potassium chloride, KCI, also known as potassium muriate and sylvite, is a colorless crystalline solid with a salty taste that melts at 776°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