2. The supernatants are collected and centrifuged at 3000 Â g for
10 min in a Beckman Coulter Allegra X-22 Centrifuge. The
supernatants are then filtered through a 0.22-μm filter and
added to new ultracentrifuge tubes.
3. Two serial ultracentrifuges with Hitachi CP100NX are performed. The first one is performed at 100,000 Â g for 2 h at
4
C. The pellet is then re-dissolved in 15 mL of saline solution.
Then, a second centrifugation is done at 100,000 Â g for 2 h at
4
C, and the last pellet is re-dissolved in 100 μL of lysis buffer
or RIPA buffer for protein extraction.
3.2 Particle Number
Characterization by
Nanoparticle Tracking
Analysis (NTA)
The NanoSight LM10 is calibrated using silica microsphere beads.
MSC-derived EVs are diluted in PBS in order to obtain a particle
number ranging from 10
8 to 10
9 particles/μL. At least three
repeated measurements of 60 μs are taken per each individual
sample, and the mean value is used to determine particle number
(Fig. 1a). The movement of each particle in the field of view is
measured to generate the average displacement of each particle per
unit time, which is calculated using the NTA 3.0.
3.3 Morphology
Characterization by
Transmission Electron
Microscopy (TEM)
1. Re-suspend the EVs in 100 μL of 2.5% glutaraldehyde in 0.1 M
cacodylate buffer. This fixation method is the most appropriate
for EV observation by TEM to avoid salt precipitation from the
medium.
2. Apply the EV suspensions in a metal grid and air-dry.
3. Observe in a JEOL JEM-1010 (100 kV) transmission electron
microscope (Fig. 1b).
3.4 Protein
Extraction and
Quantification
1. Incubate the pellet containing the EVs, which was
re-suspended in 100 μL of RIPA buffer, in rotation for
30 min at 4
C. Add 1% protease inhibitor cocktail immediately
before use.
2. Centrifuge at 14,000 Â g for 20 min at 4
C.
3. Add the supernatant to a new tube to which six volumes of
acetone was added and precipitate at À20
C for at least 3 h
with sporadic gentle shaking (see Note 1).
4. Centrifuge at 14,000 Â g for 10 min at 4
C.
5. Carefully decant supernatant and briefly air-dry the pellet. Add
100 μL of 0.1 M TEAB.
6. Incubate the pellet in a thermoblock at 25
C with shaking at
1400 rpm for 5 min.
7. Sonicate the pellet for 5 min. Repeat steps 6 and 7 until the
pellet is totally dissolved (see Note 2).
6
Miriam Morente-Lo ´ pez et al.
10 min in a Beckman Coulter Allegra X-22 Centrifuge. The
supernatants are then filtered through a 0.22-μm filter and
added to new ultracentrifuge tubes.
3. Two serial ultracentrifuges with Hitachi CP100NX are performed. The first one is performed at 100,000 Â g for 2 h at
4
C. The pellet is then re-dissolved in 15 mL of saline solution.
Then, a second centrifugation is done at 100,000 Â g for 2 h at
4
C, and the last pellet is re-dissolved in 100 μL of lysis buffer
or RIPA buffer for protein extraction.
3.2 Particle Number
Characterization by
Nanoparticle Tracking
Analysis (NTA)
The NanoSight LM10 is calibrated using silica microsphere beads.
MSC-derived EVs are diluted in PBS in order to obtain a particle
number ranging from 10
8 to 10
9 particles/μL. At least three
repeated measurements of 60 μs are taken per each individual
sample, and the mean value is used to determine particle number
(Fig. 1a). The movement of each particle in the field of view is
measured to generate the average displacement of each particle per
unit time, which is calculated using the NTA 3.0.
3.3 Morphology
Characterization by
Transmission Electron
Microscopy (TEM)
1. Re-suspend the EVs in 100 μL of 2.5% glutaraldehyde in 0.1 M
cacodylate buffer. This fixation method is the most appropriate
for EV observation by TEM to avoid salt precipitation from the
medium.
2. Apply the EV suspensions in a metal grid and air-dry.
3. Observe in a JEOL JEM-1010 (100 kV) transmission electron
microscope (Fig. 1b).
3.4 Protein
Extraction and
Quantification
1. Incubate the pellet containing the EVs, which was
re-suspended in 100 μL of RIPA buffer, in rotation for
30 min at 4
C. Add 1% protease inhibitor cocktail immediately
before use.
2. Centrifuge at 14,000 Â g for 20 min at 4
C.
3. Add the supernatant to a new tube to which six volumes of
acetone was added and precipitate at À20
C for at least 3 h
with sporadic gentle shaking (see Note 1).
4. Centrifuge at 14,000 Â g for 10 min at 4
C.
5. Carefully decant supernatant and briefly air-dry the pellet. Add
100 μL of 0.1 M TEAB.
6. Incubate the pellet in a thermoblock at 25
C with shaking at
1400 rpm for 5 min.
7. Sonicate the pellet for 5 min. Repeat steps 6 and 7 until the
pellet is totally dissolved (see Note 2).
6
Miriam Morente-Lo ´ pez et al.