2 Materials
All reagents were prepared using Milli-Q water (obtained through
purifying the deionized water to attain a resistance of 18 MΩ cm at
25
C) and filtered through 0.22 μm (sterilization) prior to use. All
biological wastes were discarded into the biohazard bag and disposed in accordance with the proper waste disposal guideline.
2.1 Cell Culture
1. Human pro-monocytic myeloid leukemia cell line (U937) (see
Note 1).
2. Complete culture medium: RPMI-1640 medium supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS).
3. Sterile 50 and 15 mL tubes.
4. Protease inhibitor cocktail (EDTA-free,): AEBSF hydrochloride (100 mM), aprotinin (bovine lung) (0.08 mM), bestatin:
(5 mM), E-64 (1.5 mM), leupeptin hemisulfate (2 mM), pepstatin A (1 mM).
5. Phosphate-Buffered Saline (PBS): adjusted to pH 7.4 with
hydrochloric acid (HCl), sterilized.
6. 0.4% Trypan Blue solution: prepared in PBS.
7. Light microscope.
8. Hemocytometer (cell counting chamber).
2.2 Centrifugation
1. Benchtop refrigerated microcentrifuge machine with the
capacity of reaching 14,000 Â g.
2. Spin cups supplied with 10 μm filters attached.
3. 8 μm Hydrophilic polycarbonate membranes.
4. Hole punctures (hole diameter: 9 mm).
2.3 Size Exclusion
Chromatography (SEC)
1. Sephadex G-50 self-packed size exclusion column.
2. PBS: Ice-cold, pH 7.4.
3. Sterile 1.5 mL microcentrifuge tubes.
2.4 Physical
Characterization
2.4.1 Hydrodynamic Size
and Zeta Potential
Measurement
1. CDNs samples.
2. Zetasizer Nano (Malvern Instruments).
3. Folded capillary cell/polycarbonate cell with gold-plated
electrodes.
4. Disposable polystyrene cuvette.
5. De-ionized (D.I.) water.
6. Kimwipes.
160
Yi-Hsuan Ou et al.
All reagents were prepared using Milli-Q water (obtained through
purifying the deionized water to attain a resistance of 18 MΩ cm at
25
C) and filtered through 0.22 μm (sterilization) prior to use. All
biological wastes were discarded into the biohazard bag and disposed in accordance with the proper waste disposal guideline.
2.1 Cell Culture
1. Human pro-monocytic myeloid leukemia cell line (U937) (see
Note 1).
2. Complete culture medium: RPMI-1640 medium supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS).
3. Sterile 50 and 15 mL tubes.
4. Protease inhibitor cocktail (EDTA-free,): AEBSF hydrochloride (100 mM), aprotinin (bovine lung) (0.08 mM), bestatin:
(5 mM), E-64 (1.5 mM), leupeptin hemisulfate (2 mM), pepstatin A (1 mM).
5. Phosphate-Buffered Saline (PBS): adjusted to pH 7.4 with
hydrochloric acid (HCl), sterilized.
6. 0.4% Trypan Blue solution: prepared in PBS.
7. Light microscope.
8. Hemocytometer (cell counting chamber).
2.2 Centrifugation
1. Benchtop refrigerated microcentrifuge machine with the
capacity of reaching 14,000 Â g.
2. Spin cups supplied with 10 μm filters attached.
3. 8 μm Hydrophilic polycarbonate membranes.
4. Hole punctures (hole diameter: 9 mm).
2.3 Size Exclusion
Chromatography (SEC)
1. Sephadex G-50 self-packed size exclusion column.
2. PBS: Ice-cold, pH 7.4.
3. Sterile 1.5 mL microcentrifuge tubes.
2.4 Physical
Characterization
2.4.1 Hydrodynamic Size
and Zeta Potential
Measurement
1. CDNs samples.
2. Zetasizer Nano (Malvern Instruments).
3. Folded capillary cell/polycarbonate cell with gold-plated
electrodes.
4. Disposable polystyrene cuvette.
5. De-ionized (D.I.) water.
6. Kimwipes.
160
Yi-Hsuan Ou et al.