Isolation of EVs from brain parenchyma

Isolation of extracellular vesicles from cell culture conditioned medium

• 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 in serum-free OptiMEM (preferrable) or in basic DMEM supplemented with 10% FBS + P/S + Glutamax previously depleted of Extracellular vesicles (EVs) by centrifugation at 100,000g for 16h. 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 1.5 mL tubes, Beckmann 45Ti for 70 ml tubes). 
• Sterile pipettor and pipettes.
• Ice cold glass vessel + pestle.
• Beckman Coulter #357448 1.5 mL tubes. Do not use standard 1.5 mL tubes!

Material/Solutions

• Cells grown in EV-depleted medium.
• PBS kept at 4 °C.
• Buffer A: 100 mM KCl, 50 mM Tris, 5 mM MgCl2, 1.8 mM ATP, and 1 mM EDTA adjusted to pH 7.2. Make larger volumes and freeze it at -20ºC for long term storage. 

Notes

• All centrifugation steps are conducted at 4 ºC.
• Always keep your samples in ice (plates, EVs etc.) to avoid 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).
• EV isolation from cell culture conditioned medium yields low quanta of EVs, making the EV 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 EV-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.
• 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 degrade in ice.

PROTOCOL

PART A: 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. 
• 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 polypropylene microfuge tube (e.g., Beckman Coulter #357448). Optional: it’s better to resuspend the pellet in two different steps, 500 µL 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 EV pellet in PBS.

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

• Wash the 10 plates containing cells 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. A high 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.
• Collect the homogenate and separate a small aliquot (‘Total Lysate’).
• Centrifuge 800g for 5 min. 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. The pellet will contain mitochondria and other heavy membranous organelles (lysosomes, peroxisomes, some Golgi, some ER etc.).
• Wash the pellet with 1 mL Buffer A and centrifuge again 15.000g for 10 min.
• Resuspend the crude mitochondrial pellet in Buffer A.

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