mtDNA extraction from Fr8 EVs and qPCR

Isolation of extracellular vesicles from cell culture conditioned medium and analysis of ATP production

(Levy lab, ATP production assay originally adapted from Drew and Leeuwenburgh, 2003, and integrated with Lanza and Nair, 2009, modified in some parts by D'Acunzo P., 2018)

• 2 days before the isolation: split the cells from 100% confluent population grown in complete medium into different plates so they reach 70% confluence the following day
• 1 day before the isolation: Wash the cells once with PBS and incubate in basic DMEM supplemented with 10% FBS + P/S + Glutamax previously depleted of Extracellular vesicles (EVs) by centrifugation at 100,000g for 16h or in serum-free OptiMEM. This protocol has been set up for ten 100 mm culture dish plates (6mL of medium each plate)

Equipment set-up

• Bench-top centrifuge chamber cooled at 4 °C
• High speed centrifugation polycarbonate tubes
• High speed centrifuge chamber with designated rotor cooled at 4°C and under vacuum (Beckmann MLA-80 rotor for 8 ml tubes, TLA-55 for Eppendorf tubes, Beckmann 45Ti for 70 ml tubes)
• Sterile pipettor and pipettes
• Ice cold glass vessel + pestle
• Flat, untreated, white, polystyrene plates for SpectraMax (do not use black or transparent ones used for BCA). The top-quality ones are the MicroFluor 1, White (ThermoFisher) and the 3912, Corning White (Corning)

Material/Solutions

• Cells grown in EV-depleted medium
• PBS kept at 4 °C
• ATP detection Kit (code A22066, ThermoFisher)
• Buffer A: 100 mM KCl, 50 mM Tris, 5 mM MgCl2, 1.8 mM ATP, and 1 mM EDTA adjusted to pH 7.2. You can make larger volumes and Freeze it at -20ºC for long term storage. ATP degrades fast, don’t put it at 4 °C
• Buffer B:
  - For short term storage (1-2hs): 225 mM sucrose, 44 mM KH₂PO₄, 12.5 mM Mg-Acetate, 6 mM EDTA. It’s the one usually used in this protocol
  - For up to overnight storage of mitochondria at 4°C: 0.5 mM EGTA, 3mM MgCl2*6H2O, 60 mM potassium lactobionate, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM Sucrose, 0.1% fatty acid free BSA, 20 mM histidine, 20 μM vitamin E succinate, 3 mM glutathione, 1 μM leupeptine, 2 mM glutamate, 2 mM malate, 2 mM Mg-ATP (as reported by Lanza and Nair, 2009). This buffer simulates better the cytosol, and is appropriate for long term storage of intracellular mitochondria

Notes

• All centrifugation steps are conducted at 4 ºC
• Always keep your samples in ice (plates, EVs etc.) to avoid mitochondria degradation
• Always fill high centrifuge tubes up to at least ¾ of the total volume
• For ultracentrifugation steps in a fixed angle rotor, mark the side of the tube facing up in the centrifuge rotor to point out the location of the pellet.
• Make sure that all high speed centrifuge tubes have the same weight (to the 0.01g).
• Exosome isolation with cell culture conditioned medium yields drastically lower quanta of exosomes than fresh brain tissue making the exosome pellet difficult to spot and to recover in a large volume high speed centrifugation tube (you cannot see it). For this reason, it is best to grow cell cultures in the smallest volume of exosome-free medium possible (up to 6ml in a 100 mm culture dish, so that 10 plates will fit into a 70 mL tube).
• Remember to close the centrifuges at least half an hour before starting! This way they will be cooled
• Mitochondria functionality is extremely sensitive. Do not over homogenize as this will break mitochondria outer membrane. Do not use PBS after homogenization and try to be as fast as possible since mitochondria lose activity overtime in ice

PROTOCOL

PART 1A: ISOLATE EVs FROM CELL MEDIA

• Collect the conditioned media from the cell in EV-free medium in two 50 ml conical Falcon tube (30 mL each).
• Centrifuge the tubes at 300g for 10 min at 4ºC
• Transfer the supernatant to another two 50 mL conical tubes (leave a bit of liquid to avoid touching the pellet). Centrifuge at 2,000g for 10 minutes at 4°C.
• Collect and transfer both supernatants in one 70 mL polycarbonate Beckman tube
• Centrifuge the tube for 30 minutes at 10,000g (11,000 rpm in a 45Ti Rotor) at 4 ºC.
• Pipette the supernatant carefully to another Beckman tube leaving the debris pellet undisturbed. Leave enough supernatant in the tube so that the pellet stays submerged and does not contaminate the supernatant containing the exosomes.
• Load the 45Ti Rotor again
• Centrifuge the tube for 70 min at 100,000g (36,000 rpm in a Ti45 rotor) at 4 ºC.
• Discard the supernatant without disturbing the EV pellet (for a fixed angle rotor, the supernatant can be poured off)
• Resuspend the pellet in 1 ml of PBS kept at 4 ºC and transfer to a microfuge tube polypropylene (Beckman Coulter #357448). DO NOT USE STANDARD TUBE AS THEY WILL BREAK! Optional: it’s better to make two different steps, 500 uL each, to take all the EVs
• Centrifuge the tube for 70 minutes at 100,000g (50,000 rpm for TLA-55 rotor) at 4 ºC. Remember to ‘click’ the button on the rotor otherwise it will not be locked!
• Pipette the supernatant without disturbing the EV pellet. Keep the pellet at 4 ºC.
• Resuspend the pellet into Buffer B and quickly go on with the ATP production experiment (PART2).

PART 1B: ISOLATE A CRUDE MITOCHONDRIAL PELLET FROM CELLS AS A POSITIVE CONTROL (TO BE DONE WHILE EXTRACTING EVs)

• Wash the 10 plates with PBS to remove any media remnants.
• Add 1 mL of ice-cold PBS to each plate and gently detach cells using a cell scraper
• Collect the suspensions and centrifuge 300g for 5 min to collect cells
• Discard the supernatant and weight the pellets
• Add 1:10 w/v of Buffer A, resuspend the pellets and quickly move the suspension the cold glass vessel to be used for homogenization
• Homogenize in ice using 20 complete up-and-down strokes of the pestle. This step is critical for mitochondrial integrity, since a high fractional yield typically requires harsh homogenization methods that often damage the organelles, while, gentle homogenizing procedures preserves the integrity of the outer membrane at the expense of lower yield. Follow these rules:
          - Annular flow shear forces destroy mitochondrial outer membrane      (Rasmussen et al., 1997a), so use gentle, regular and slow vertical plunger
    movements. Stay in the range of 30 seconds per single stroke (10 min total time)

  - Do not use motor-driven homogenizers

  - Remember to make planar circular movements while moving up and down
  - Pause for few seconds the up and down movement but not the circular one when
  reaching the bottom of the vessel, in order to complete break heavy clumps

• Collect the homogenate and separate a small aliquot (‘Total Lysate’) if a Western Blot follows (as a positive control). Centrifuge 800g for 5 min (3000 rpm into the TX-160 centrifuge into the Levy lab). The pellet will contain nuclei and unbroken cells. Save the supernatant and repeat this step to be sure no pellet will be carried on
• Centrifuge the supernatant at 15.000g for 10 min (max speed with our TX-160 benchtop centrifuge). The pellet will contain mitochondria and other heavy membranous organelles (lysosomes, peroxisomes, some Golgi, some ER etc.). The supernatant will contain microsomes (vesicles of various origins, pieces of ER, Golgi, plasma membrane etc.) and the cytosol. Save the pellet and a small aliquot of the cytosol as a negative control
• Wash the pellet with 1 mL Buffer A and centrifuge again 15.000g for 10 min
• Resuspend the crude mitochondrial pellet into Buffer B. Use 4 ul of buffer B for each milligram of cell pellet (see point 4 of this section). After resuspension, mitochondrial ATP activity is stable in ice for up to 1h then starts to decrease (as reported in Lanza and Nair, 2009), so be fast!

PART 2: MEASURING MITOCHONDRIAL ATP

1. Measure protein concentration through high-sensitive BCA protocol of mitochondrial, cytosolic (control) and EVs preps. The ATP assay is sensitive up to 4 ug of total pure mitochondrial proteins with an integrity of 60%, and 10 ug of EVs
2. Prepare ATP standards in water: 10 nM. 50 nM, 100 nM, 500 nM, 1 µM. Standards can be frozen at -20 ºC.
3. Prepare stocks of the MasterMix Solution and of FCCP and OA, according to Table 1. Stocks can be saved at -20 ºC for several weeks.
4. Mix the components of the MasterMix solution according to Table 2. Adjust the final volume of the MasterMix taking into account that each well of the assay will contain 200 ul of Mix and 10 ul of sample. For each experiment, load 2 blanks and 5 standards in duplicate + 6 samples per condition to be tested (untreated, +OA and +FCCP in duplicate).
5. Aliquot the MasterMix to be added with FCCP (0.85 ml for two conditions in duplicate) and with Oligomycin and Antymicin-A (0.85 ml for two conditions in duplicate). Dilute FCCP and OA to 1X, respectively.
6. Load the plate with 200 ul of MasterMix per well, add 10 ul of samples or standards in each well (10 ul of water in the blank) and read in the microplate reader for 1h every 2 minutes at 37 ºC.

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