Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification

Gloves must be worn at all times and cell culture steps performed under a laminar flow hood. The cells are maintained in a 37 °C incubator with 5% CO₂. The research presented in this protocol was approved and performed in compliance with the University of Lausanne and Swiss guidelines for the use of animals.

1. Culture of RAW264.7 macrophage cell line

1. Grow the mice macrophage RAW264.7 cells in Dulbecco's modified eagle medium (DMEM) with high glucose and glutamine supplemented with 5% heat-inactivated fetal bovine serum (HI-FBS) and 100 IU/mL penicillin and 100 µg/mL streptomycin (P/S).
   NOTE: To isolate mitochondria, a single confluent 15 cm plate (about 70 x 10⁷ RAW264.7 cells) is sufficient.
2. Maintain the RAW264.7 cells in tissue culture plates. An initial seeding density of 1 x 10⁵ cells/mL leads to a confluent plate in 3 days. Use 25 mL of cell suspension in media for a 15 cm plate. RAW264.7 cells have a high cell division rate and need to be split more frequently than most cell lines.
3. Detaching RAW264.7 cells
   1. Aspirate the media and wash the cells once with phosphate-buffered saline (PBS).
   2. For a 15 cm plate, add 8 mL of warm RAW dissociation buffer (270 mM potassium chloride, 30 mM sodium citrate dihydrate in H₂O, sterile filtered) and incubate the cells at 37 °C for 5 min.
   3. Add an equivalent volume of media to the plates (1:1 dilution of the dissociation buffer), and pipette to detach and homogenize the cells.
   4. Transfer the cell suspension to a conical tube and centrifuge the tube at 300 x g for 3 min at room temperature.
   5. Aspirate the supernatant and resuspend the pellet in an appropriate volume of media (described in step 1.1) for cell counting.
      ​NOTE: Other methods to detach RAW264.7 cells can be used, such as trypsin or a cell scraper. However, these methods are harsher on the cells and may lead to their polarization into M1-like macrophages in the days following detaching.

2. Isolation and culture of bone marrow-derived macrophages (BMDMs)

NOTE: The protocol described here is for a single mouse and can be scaled up for multiple mice. Detailed protocols for BMDM isolation and culture have been described elsewhere^13,14.

1. Sacrifice an 8-12-week-old C57BL/6 mouse with a high dose of CO₂.
   NOTE: Either male or female mice can be used.
2. Spray the mouse with 75% ethanol to sterilize it.
3. Dissect and collect the hips, femurs, and tibias from the mouse¹⁵.
4. To collect the bone marrow from the femurs and tibias, remove the knee joint end of both bones¹⁵. Recover bone marrow from the hips by removing the acetabulum.
5. Transfer the bones to a 50 mL conical tube with 4 mL of BMDM media kept on ice (DMEM with high glucose and glutamine supplemented with 5% HI-FBS, P/S, and 10 mM HEPES).
   NOTE: It is important to keep the bones in the media to avoid drying of the bone marrow during dissection.
6. Add 4 mL of PBS and 4 mL of warm BMDM media in two different wells of a 6-well plate.
7. Transfer the bones and the media from the 50 mL conical tube to an empty well of the 6-well plate.
8. Using a pair of forceps, transfer the bones to the PBS well to wash them.
9. Transfer the bones to the well containing warm BMDM media.
10. Make a hole of 1-2 mm in diameter in the bottom of two 0.5 mL tubes with the forceps and place them in a 1.5 mL tube.
    NOTE: It is not necessary to add media to the tube for this quick step.
11. In each 0.5 mL tube, place a femur, tibia, and hip in such a way that the bone marrow of the exposed bones faces toward the bottom of the tubes.
12. Centrifuge the tubes at 13,000 x g for 1 min at room temperature to collect the bone marrow and leftover media through the hole of the 0.5 mL tube and into the 1.5 mL tube. Discard the 0.5 mL tubes with the bones.
13. Resuspend the bone marrow pellets in BMDM media and transfer to a 15 mL conical tube.
14. Add BMDM media up to 10 mL.
15. Place a 40 µm cell strainer on a 50 mL conical tube and filter the bone marrow suspension through it.
16. Centrifuge the filtered suspension at 300 x g for 5 min at room temperature to recover the intact cells and remove small debris from the cell suspension.
17. Prepare 70 mL of BMDM media supplemented with 50 ng/mL of macrophage colony-stimulating factor (M-CSF).
18. Resuspend the pellet in 10 mL of M-CSF-supplemented BMDM media.
19. Add 9 mL of M-CSF-supplemented BMDM media to each of the seven 10 cm Petri dishes.
20. Plate 1 mL of cells (around 1 x 10⁷ cells) in each of the seven 10 cm Petri dishes.
21. Homogenize the cell suspension in each plate by carefully pipetting up and down on the plate and transfer the plates to the incubator.
22. After 3 days, add 5 mL of warm BMDM media supplemented with 50 ng/mL M-CSF to each plate.
23. After 3 days (day 6, after bone marrow isolation), verify adhesion and differentiation of the BMDM by microscopy.
    NOTE: At this point, it is possible to proceed directly to mitochondria isolation (step 3). Alternatively, one can replate the BMDM, which allows treating them with cytokines and small molecules.
24. Detaching the BMDMs
    1. Aspirate the media from each plate and add 7 mL of cold PBS supplemented with 5 mM ethylenediaminetetraacetic acid (EDTA).
    2. Incubate the cells at 4 °C for 7-8 min.
    3. Detach the BMDMs by carefully pipetting up and down using a 10 mL pipette.
    4. Pool together the resuspended BMDMs from all seven plates into a single 50 mL conical tube and centrifuge at 300 x g for 3 min at room temperature.
    5. Aspirate the supernatant and resuspend the cells in 40 mL of warm BMDM media for cell counting.
       NOTE: Between 7-9 x 10⁷ BMDMs are obtained per mouse. A minimum of 6 x 10⁷ BMDMs for mitochondria isolation is recommended for proteomics.
    6. If desired, plate and treat the BMDMs according to the experimental goal. If not, proceed directly to step 3.3.

3. Preparation of a crude mitochondrial fraction by differential centrifugation

NOTE: Perform all centrifugation steps at 4 °C. Two centrifuges are required, one with a swing-out rotor and adaptors for conical tubes with a relative centrifugal force of at least 300 x g, the other with a relative centrifugal force of at least 21,000 x g suitable for 1.5 mL tubes. When using adherent cells, use a cell scraper.

1. For adherent cells, aspirate the media and add 10 mL of PBS per 15 cm plate.
   NOTE: Scraping cells in PBS allows one to wash them at the same time. If the cells are already in the suspension, proceed directly to step 3.3.
2. Detach the cells using a cell scraper and pool them in a single 50 mL conical tube. Homogenize the cell suspension by pipetting up and down.
   NOTE: Cells can be detached using a cell scraper, since this is faster and they will be lysed shortly after.
3. For each experimental condition, transfer 5% of the cell suspension volume to a 1.5 mL tube and centrifuge it at 300 x g for 5 min at room temperature.
   NOTE: When using suspension cells, make sure to wash the cells once with PBS before, to remove possible contaminants from the media such as FBS.
4. Discard the supernatant and keep the pellet on ice.
   NOTE: This will represent the "total cell" fraction for proteomics.
5. Centrifuge the rest of the samples from step 3.1 or 3.2 at 300 x g for 5 min at room temperature.
6. Perform all following steps on ice and using ice-cold buffers.
7. Aspirate the supernatant and resuspend the cell pellet in 5 mL of ice-cold mitochondria buffer (MB) (210 mM mannitol, 70 mM sucrose, 10 mM HEPES/NaOH [pH 7.4], and 1 mM EDTA).
8. Recover the cells by centrifugation at 300 x g for 5 min at 4 °C.
9. Resuspend the cell pellet in 0.5 mL of cold MB and transfer it to a 1.5 mL tube.
   NOTE: This procedure yields an approximate cell concentration of 1.5 x 10⁸ BMDM cells/mL or 3 x 10⁸ RAW264.7 cells/mL.
10. Using a 1 mL syringe fitted with a 25 G needle, homogenize the cell suspension by 30 passages through the needle (Figure 1A).
11. Add 1 mL of cold MB to the tube and mix by inversion. Centrifuge the homogenized cell suspension at 2,000 x g for 5 min at 4 °C.
12. Transfer 1 mL of the supernatant to a fresh 1.5 mL tube on ice without disturbing the cell pellet. Resuspend the cell pellet and homogenize it again, as in step 3.7.
13. Pool the homogenized cell pellet and the supernatant from the two previous steps in a single 1.5 mL tube and centrifuge it at 2,000 x g for 5 min at 4 °C.
    NOTE: At this point, the pellet contains mostly nuclei and unbroken cells and is discarded. The supernatant contains cellular debris, cytosol, and organelles, including mitochondria (Figure 1B).
14. Divide the supernatant among four 1.5 mL tubes.
    NOTE: Dividing the supernatant among multiple tubes at this stage improves the removal of contaminants in the following steps.
15. Add MB to make a final volume of 1 mL in each of the four tubes. Mix by inversion and centrifuge the tubes at 13,000 x g for 10 min at 4 °C.
    NOTE: After this step, a pellet with two layers is visible. The bottom, firm, brown pellet contains mitochondria and is kept for further purification (Figure 1C). The upper, loose, white pellet contains other cellular structures and can be discarded.
16. Perform this step carefully. Remove the supernatant with as much of the white upper pellet as possible. By gently pipetting on it, it is possible to resuspend the white pellet and then discard it, leaving the brown mitochondrial pellet intact.
17. Keep one of the four tubes with the mitochondrial pellet on ice. This represents the "crude mitochondria" fraction for proteomics.
18. Pool together the other three pellets in a 1.5 mL tube in a final volume of 1 mL of MB.

4. Superparamagnetic antibody-based purification of mitochondria

NOTE: Perform all the following steps in a cold room at 4 °C.

1. Transfer 1 mL of the crude mitochondrial preparation from step 3.18 to a 15 mL conical tube and add 7 mL of MB supplemented with 150 mM NaCl (MB + NaCl).
   NOTE: The addition of NaCl improves antibody binding and decreases non-specific binding of the contaminants to the beads and to mitochondria.
2. Add 50 µL of Tomm22 beads to the 8 mL crude mitochondria suspension (Figure 1D) and incubate the tube for 15 min at 4 °C on a rotating wheel at low speed.
   NOTE: Tomm22 beads are covalently bound to Tomm22 monoclonal antibodies raised in mouse, coupled to superparamagnetic beads.
3. Meanwhile, place a column on the magnet.
4. Equilibrate the column with 8 mL of MB + NaCl and discard the flowthrough.
5. Following the 15 min incubation at 4 °C of the sample with the Tomm22 beads, transfer the sample into the column. Discard the flowthrough.
   NOTE: Mitochondria will remain attached to the magnetic beads in the column (Figure 1E).
6. Wash the column three times with 8 mL of MB + NaCl.
7. Remove the column from the magnet and place it in a 15 mL conical tube.
8. Elute the mitochondria by adding 1.5 mL of MB + NaCl to the column and apply a plunger immediately to elute the purified mitochondria into the tube.
9. Transfer the eluted mitochondria to a 1.5 mL tube and centrifuge it at 21,000 x g for 10 min at 4 °C.
   NOTE: A brown pellet will form. This contains the isolated mitochondria and some of the antibody-coupled beads (Figure 1F).
10. Remove the supernatant carefully from the pellet. The pellet represents the "pure mitochondria" fraction for proteomics. This pellet together with the pellets from steps 3.4 and 3.17 can be stored at -20 °C and are ready for downstream applications.