could provide a molecular basis for their distinct functional
properties [6].
This protocol describes the steps to process human umbilical
cord stromal MSC-derived EVs for subsequent analyses. The focus
was the isolation of EVs from culture medium by serial ultracentrifugation and subsequent protein extraction from EVs using RIPA
buffer.
2 Materials
The cell culture procedures are performed in a class II flow cabinet.
All solutions are prepared using ultrapure water (prepared by purifying deionized water to a sensitivity of 18 MΩ cm at 25
C) and
analytical-grade reagents. All organic solvent solutions are LC
MS-grade. All reagents are prepared and stored at 4
C, and all
waste disposal regulations are diligently followed when disposing of
waste materials.
2.1 EV Extraction
from Culture Medium
by Serial
Ultracentrifugation
1. Mesenchymal stem cells in culture growing at a 70%
confluence.
2. Culture medium Dulbecco’s modified Eagle’s medium
(DMEM).
3. Exosome-depleted fetal bovine serum (FBS).
4. Penicillin–streptomycin (P/S).
5. Sodium chloride (NaCl).
6. Filters (0.22 μm).
7. Refrigerated centrifuge of ultracentrifugation of soil
100,000 Â g.
8. Culture centrifuge.
9. Ultracentrifuge tubes.
2.2 Particle Number
Characterization by
Nanoparticle Tracking
Analysis (NTA)
1. Syringe of 1-mL Luer slip, Plastipak.
2. PBS (phosphate-buffered saline).
3. Silica microsphere beads.
4. NanoSight LM10.
2.3 Morphology
Characterization by
Transmission Electron
Microscopy (TEM)
1. Sodium cacodylate (Na(CH 3 ) 2 AsO 2 ).
2. 37% hydrochloric acid (HCl).
3. pH meter GLP 21.
4. Glutaraldehyde (C 5 H 8 O 2 ).
5. JEOL JEM-1010 (100 kV) transmission electron microscope.
4
Miriam Morente-Lo ´ pez et al.
properties [6].
This protocol describes the steps to process human umbilical
cord stromal MSC-derived EVs for subsequent analyses. The focus
was the isolation of EVs from culture medium by serial ultracentrifugation and subsequent protein extraction from EVs using RIPA
buffer.
2 Materials
The cell culture procedures are performed in a class II flow cabinet.
All solutions are prepared using ultrapure water (prepared by purifying deionized water to a sensitivity of 18 MΩ cm at 25
C) and
analytical-grade reagents. All organic solvent solutions are LC
MS-grade. All reagents are prepared and stored at 4
C, and all
waste disposal regulations are diligently followed when disposing of
waste materials.
2.1 EV Extraction
from Culture Medium
by Serial
Ultracentrifugation
1. Mesenchymal stem cells in culture growing at a 70%
confluence.
2. Culture medium Dulbecco’s modified Eagle’s medium
(DMEM).
3. Exosome-depleted fetal bovine serum (FBS).
4. Penicillin–streptomycin (P/S).
5. Sodium chloride (NaCl).
6. Filters (0.22 μm).
7. Refrigerated centrifuge of ultracentrifugation of soil
100,000 Â g.
8. Culture centrifuge.
9. Ultracentrifuge tubes.
2.2 Particle Number
Characterization by
Nanoparticle Tracking
Analysis (NTA)
1. Syringe of 1-mL Luer slip, Plastipak.
2. PBS (phosphate-buffered saline).
3. Silica microsphere beads.
4. NanoSight LM10.
2.3 Morphology
Characterization by
Transmission Electron
Microscopy (TEM)
1. Sodium cacodylate (Na(CH 3 ) 2 AsO 2 ).
2. 37% hydrochloric acid (HCl).
3. pH meter GLP 21.
4. Glutaraldehyde (C 5 H 8 O 2 ).
5. JEOL JEM-1010 (100 kV) transmission electron microscope.
4
Miriam Morente-Lo ´ pez et al.