Isolation of Mitochondria from Plants, Yeast Cells, Mice, Cell Culture 5/5 (2)

Isolation of Mitochondria from Cell Culture


Isolation of Mitochondria from Plants, Yeast Cells, Mice, Cell Culture

  • Mitochondria are central 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 cascades, including apoptosis.
  • Investigating functional mitochondria that have been isolated from different tissues and from cultured cells offers a full understanding of mitochondrial function.
  • Mitochondria can be isolated from a variety of cells or tissues. 

Isolation of Mitochondria from Plants (Potato Tubers)

Isolation of Mitochondria from Plants (Potato Tubers)



Materials and Reagents

  • Mannitol
  • 3-morpholinopropane-1-sulfonic acid (MOPS)
  • Potassium hydroxide (KOH)
  • Bovine serum albumin (BSA)
  • Ethylenediaminetetraacetic acid (EDTA) 
  • Cysteine
  • Percoll
  • Sucrose
  • Dimethylsulfoxide (DMSO)
  • Liquid nitrogen
  • Extraction medium
  • Wash buffer
  • Gradient buffers
  • Percoll gradients

Equipment

  • Potato tubers
  • 6 x 250 ml precooled angle rotor
  • 8 x 50 ml pre-cooled angle rotor
  • Juice extractor
  • Centrifuge (for 50 and 250 ml tubes)
  • Paint brush (soft)

Procedure

  1. Pre-cool centrifuge rotor.
  2. Add cysteine and BSA to extraction buffer, adjust to pH 7.3. Cysteine is an antioxidant and BSA binds fatty acids and phenolics, which can interfere with mitochondrial function, BSA also works as a protease substrate to help protect the mitochondrial proteins from damage.
  3. Peel potatoes and homogenize the peeled potato using a juice extractor (1 kg gives approximately 500 ml juice) and let the juice run directly into about 1/2 volume extraction medium (250 ml here). Adjust to pH 7.2 using 2 M KOH immediately afterwards or, for larger preparations, after each kg homogenized.
  4. Leave homogenate (total volume about 750 ml) standing for 5 min allowing starch to sediment.
  5. Filter through two layers of cotton (or similar) using a funnel and transfer to centrifuge tubes.
  6. Transfer ~190 ml filtrate to each of four 250 ml centrifuge tubes. Balance tubes, and centrifuge at 3,000 x g for 5 min in a 6 x 250 ml precooled angle rotor.
  7. Pour supernatants carefully into fresh centrifuge tubes (avoid transferring the pellets), balance and centrifuge at 18,000 x g for 10 min.
  8. Discard the supernatants gently without disturbing the pellets and resuspend each pellet in 1 ml of 1x mannitol gradient buffer using a paint brush. Total volume of resuspended pellets 8-10 ml.
  9. Prepare two Percoll step gradients using plastic Pasteur pipettes. Avoid mixing the bands by gently layering first the 50%, then the 28% and finally the 20% Percoll (all in mannitol) on top of each other in 50 ml centrifuge tubes. 
  10. Gently layer the crude fraction (4-5 ml maximum per gradient) on top of the two Percoll step gradients.
  11. Balance tubes and centrifuge at 40,000 x g for 30 min using an 8 x 50 ml pre-cooled angle rotor.
  12. Transfer the mitochondrial band from each tube using a Pasteur pipette to new 50 ml tubes, fill up to 40 ml with wash buffer and mix.
  13. Balance the two tubes against each other and centrifuge at 18,000 x g for 10 min.
  14. Carefully remove supernatant, resuspend the very loose pellets with wash buffer, fill up to 40 ml with wash buffer and mix.
  15. Balance and centrifuge at 18,000 x g for 10 min.
  16. Remove supernatant and resuspend the very loose pellets using a paint brush in 1 ml 1 x mannitol buffer and gently layer on top of two 28% Percoll sucrose gradients.
  17. Balance and centrifuge at 40,000 x g for 30 min using the 8 x 50 ml rotor.
  18. Transfer the mitochondrial bands to two fresh 50 ml centrifuge tubes, fill up to 40 ml with wash buffer and mix. 
  19. Centrifuge at 18,000 x g for 10 min.
  20. Carefully remove supernatant and repeat wash (as steps 13-14) of pellets.
  21. Remove supernatant and resuspend each pellet in 500 µl wash buffer. Add 5% (v/v) DMSO for freezing of the intact organelles.
  22. Snap freeze and store aliquots of 100-200 µl in liquid nitrogen. In this way, the mitochondria maintain their intactness and respiratory function for months, if quickly thawed shortly before use.

Expected Result

The final volume from each pellet is around 800 µl containing 4-6 mg (total 8-12 mg) mitochondrial protein.

Isolation of Mitochondria from Microorganisms (Yeast Cells)

Materials Required

  • Yeast culture
  • Refrigerated Centrifuge
  • 15 ml centrifuge tubes
  • Sodium Chloride (0.9%)
  • Micropipette
  • Ice cold lysis buffer
  • Shaker
  • Mitochondria storage buffer
  • Refrigerator
  • 15 ml micro centrifuge tube

Procedure

  1. Aseptically transfer the overnight yeast culture into two 15ml centrifugation tubes.
  2. Centrifuge it at 500g for 10 minutes at 4°C.
  3. Carefully remove the supernatant without disturbing the pellet.
  4. Carefully rinse the pellet in 1ml sodium chloride (0.9%) using a micropipette.
  5. The sodium chloride from the centrifugation tube is discarded using a micropipette.
  6. Resuspend the pellet in 1ml of ice cold lysis buffer and mix well using a micropipette.
  7. Incubate it at 4°C on a shaker for 10 minutes.
  8. Centrifuge it at 1000g for 10 minutes at 4°C and carefully remove the supernatant.
  9. Resuspend the cell pellet in 1.5 ml ice cold disruption buffer and complete cell disruption by using the blunt end of a needle.
  10. Centrifuge the lysate at 1000g for 10 minutes at 4°C.
  11. Transfer the supernatant to a fresh 15mL tube and also mix the supernatant obtained from the step 7.
  12. Centrifuge it at 6000g for 10 minutes at 4°C and discard the supernatant.
  13. Wash the pellet with mitochondria storage buffer.
  14. Centrifuge it at 6000g for 20 minutes at 4°C.
  15. Re-suspend the pellet in mitochondria storage buffer and store at -20°C.

Isolation of Mitochondria from Liver Tissue (Mice)

Materials and Reagents

  1. Dounce homogenizer and pestles (A and B)
  2. Small scissors
  3. Tweezers
  • Centrifuge tubes
  • Mice
  • Potassium salts
  • Sodium salts
  • Sucrose
  • Bovine serum albumin (BSA)
  • Disodium ethylenediaminetetraacetate dihydrate (EDTA)
  • Ethyleneglycol-bis(2-aminoethylether)-N, N, N’, N’-tetraacetic acid (EGTA)
  • Dithiothreitol (DTT)
  • HEPES
  • Protease inhibitors (100x)
  • D-Mannitol
  • Magnesium chloride hexahydrate (MgCl2)
  • Potassium hydroxide (KOH)
  • Sodium hydroxide (NaOH)
  • Extraction buffer

Procedure


  1. Sacrifice mouse by cervical dislocation, immediately remove the liver and place it in the ice-cold beaker with extraction buffer.
  2. Rinse the liver by adding and removing cold fresh extraction buffer until most of the blood is removed (5-6 washes). 
  3. Mince the liver in the beaker in ice extensively using small scissors until homogeneous.
  4. Transfer the minced liver into a Dounce homogenizer and add approximately 3 ml of cold extraction buffer.
  5. With the homogenizer placed in the ice container, gently grind the tissue ten times with the pestle (looser) and another ten with another pestle (tighter). Avoiding the formation of bubbles is critical to obtain high quality mitochondria.
  6. Transfer the homogenate into a centrifuge tube and complete to 30-40 ml with fresh cold extraction buffer. Follow the differential centrifugation steps.
  7. Centrifuge 10 min at 700 x g and 4°C. Pour supernatant to a new ice-cold tube and discard the pellet containing nuclei and intact cells. 
  8. Repeat the operation centrifuging again at 700 x g for 10 min at 4°C and subsequently pouring the supernatant to a new ice-cold tube. 
  9. Centrifuge at 10,000 x g for 15 min at 4°C. Discard the supernatant and re-suspend the pellet in ice-cold extraction buffer. 
  10. Centrifuge at 10,000 x g for 15 min at 4°C, discard the supernatant and re-suspend the final pellet in the minimal possible volume (around 0.3 ml) of extraction buffer or the specific experimental buffer.
  11. After isolation, protein concentration is determined by standard methods.

Expected Result

Typically, around 30-40 mg of mitochondrial protein are obtained from one liver.

Isolation of Mitochondria from Skeletal Muscles (Mouse)

Reagents

  • Buffer 1 for muscle mitochondria isolation (IBm1) : Prepare 100 ml of IBm1 by mixing 6.7 ml of 1 M sucrose, 5 ml of 1 M Tris/HCl, 5 ml of 1 M KCl, 1 ml of 1 M EDTA and 2 ml of 10% BSA. Adjust pH to 7.4. Bring the volume to 100 ml with distilled water.
  • Buffer 2 for muscle mitochondria isolation (IBm2): Prepare 100 ml of IBm12 by mixing 25 ml of 1 M sucrose, 3 ml of 0.1 M EGTA/Tris and 1 ml of 1 M Tris/HCl. Adjust pH to 7.4. Bring the volume to 100 ml with distilled water.

Procedure

  1. Kill the mouse by cervical dislocation. Using a scalpel, rapidly remove the skeletal muscles of interest and immerse them in a small beaker containing 5 ml of ice-cold Phosphate buffer saline (PBS) supplemented with 10 mM EDTA.
  2. Mince the muscles into small pieces using scissors and trim visible fat, ligaments and connective tissue.
  3. Wash the minced muscles twice or thrice with ice-cold PBS supplemented with 10 mM EDTA.
  4. Resuspend the minced muscles in 5 ml of ice-cold PBS supplemented with 10 mM EDTA and 0.05% trypsin for 30 min.
  5. Centrifuge at 200g for 5 min and discard the supernatant.
  6. Resuspend the pellet in IBm1.
  7. Homogenize the muscles using a Teflon pestle operated at 1,600 r.p.m.; stroke the minced muscle ten times.
  8. Transfer the homogenate to a 50 ml polypropylene Falcon tube and centrifuge at 700g for 10 min at 4°C.
  9. Transfer the supernatant to glass centrifuge tubes and centrifuge at 8,000g for 10 min at 4 °C.
  10. Discard the supernatant and resuspend the pellet in 5 ml of ice-cold IBm2.
  11. Centrifuge at 8,000g for 10 min at 4°C.
  12. Discard the supernatant and resuspend the pellet containing mitochondria. You can use a glass rod to loosen the pellet paste. Avoid adding IB and try to resuspend the mitochondria in the small amount of buffer that remains after discarding the supernatant. Use a 200 ml pipettor and avoid the formation of bubbles during the resuspension process.
  13. Transfer mitochondrial suspension into a 14 ml tube and keep it on ice.
  14. Measure mitochondrial concentration using the Biuret methods.

Isolation of Mitochondria from Cell Culture

Isolation of Mitochondria from Cell Culture

Reagent Required

  • 1 litre cold STE with the following constituents:
    • 250 mM sucrose = 85.58g/l
    • 5 mM Tris = 0.606 g/l
    • 2 mM EGTA = 0.76 g/l
    • use 4˚C milliQ water, pH to 7.4 using 2 M HCl, keep on ice

Procedure

  1. Grow cells to 85-95% confluence in 3 x 500 cm2 tissue culture plates
  2. On growth, aspirate off all medium.
  3. Wash the cell monolayer with 30 ml cold STE, aspirate all STE off
  4. Wash for a second time with 30 ml cold STE and aspirate
  5. Scrape the cell monolayer from the plate surface using a clean razor blade
  6. Resuspend the scraped cells in 5 ml cold STE and transfer to a centrifuge tube (50ml). Repeat twice.
  7. Repeat steps 2-6 for all the tissue culture plates, pooling the cells into centrifuge tubes (1 tube per 1 tray).
  8. Spin the cells for 10 min at 4˚C 2000 rpm .
  9. Carefully aspirate the supernatant (the pellet is slightly diffuse) and retain the cell pellet.
  10. Resuspend cell pellets in 3.5 ml cold STE containing protease inhibitor and 0.5% fatty-acid-free BSA transfer to cold 7ml glass-teflon homogeniser. Wash tubes with 1.5 ml the STE with 0.5% fatty-acid-free BSA and transfer to the homogeniser.
  11. Homogenise the cells by 10 slow passes of the “tight” plunger and transfer the homogenate into a 50 ml centrifuge tube. Top up with ice cold STE if needed.
  12. Spin the homogenate at 3000 rpm for 3 min at 4˚C (1 tube)
  13. Strain the supernatant through a double layer of wet muslin and spin at 10000 rpm for 11 min at 4˚C (2 tubes)
  14. Decant supernatant and wipe inside wall of the tubes.
  15. Resuspend the mitochondrial pellet in ice cold STE, and transfer into a 15ml centrifuge tube. Top up with ice cold STE and spin at 11,600 G for 10 minutes.
  16. Re-suspend the mitochondrial pellet in residual supernatant using a cold test tube as a pestle.
  17. Keep mitochondria on ice.

Expected Result

Expected yield from 3 x 500 cm2 85% confluent plates of cells is about 15 – 25 mg of mitochondrial protein in 400 – 600 ul.



References

  1. https://bio-protocol.org/e1809
  2. Frezza, C., Cipolat, S. and Scorrano, L. (2007). Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts.Nat Protoc 2(2): 287-295.
  3. Amigo, I., Traba, J. and Rueda, C. B. (2016). Isolating Liver Mitochondria by Differential Centrifugation. Bio-protocol 6(10): e1809. 
  4. Rueda, C. B., Traba, J., Amigo, I., Llorente-Folch, I., Gonzalez-Sanchez, P., Pardo, B., Esteban, J. A., del Arco, A. and Satrustegui, J. (2015). Mitochondrial ATP-Mg/Pi carrier SCaMC-3/Slc25a23 counteracts PARP-1-dependent fall in mitochondrial ATP caused by excitotoxic insults in neurons.J Neurosci 35(8): 3566-3581.
  5. https://www.researchgate.net/profile/Irina_Perevoshchikova/post/How_can_I_isolate_mitochondria_from_cells/attachment/59d6280ec49f478072e9b900/AS%3A272429455216661%401441963651869/download/B2+mito+isolation+2+%281%29.docx
  6. https://www.nature.com/articles/nprot.2006.478
  7. https://idp.nature.com/authorize?response_type=cookie&client_id=grover&redirect_uri=https%3A%2F%2Fwww.nature.com%2Farticles%2Fnprot.2006.478
  8. http://vlab.amrita.edu/?sub=3&brch=187&sim=327&cnt=2
  9. https://bio-protocol.org/e1226

Isolation of Mitochondria from Plants, Yeast Cells, Mice, Cell Culture

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