4. Divide the 1 mL cell suspension with 2 Â 10
7 cells into 5 aliquots (200 μL/each).
5. Place each aliquot in a spin cup fitted with a 10 μm membrane
filter and centrifuge at 14,000 Â g, 4
C for 10 min. (see Notes
3 and 4).
6. Re-introduce the flow-through to the same spin cup and repeat
the centrifugation step (step 5).
7. Pipette the flow-through to another spin cup fitted with two
8 μm membrane filters and centrifuged 14,000 Â g for 10 min.
8. Re-introduce the flow-through to the same spin cup with 8 μm
membrane filters and centrifuge 14,000 Â g for 10 min.
9. Collect the flow-through of each spin cup (see Note 11).
3.3 Purification
Process
1. Pool the flow-through from five different spin cups to obtain
1 mL of the raw CDNs sample.
2. Equilibrate the Sephadex G-50 Size Exclusion Column (SEC)
with ice-cold PBS.
3. Add the raw CDNs onto SEC slowly to prevent disturbance of
the Sephadex G-50 beads.
4. Collect each 0.5 mL (one fraction) in a new 1.5 mL microcentrifuge tube. Fraction 3 and 4 are CDNs (see Note 12).
5. Pool the fractions (fraction 3 and 4) and store on ice temporarily for the characterization and further processings or at 4
C
for up to 3 days (see Note 13).
3.4 Characterization
3.4.1 Hydrodynamic Size
Measurement
The following protocol described the steps of using a Malvern
(Nano series) instruments. The protocol may need slight modifications for the application in other systems. The Zetasizer Nano
instrument should be switched on for at least 30 min before the
measurement to allow the laser to warm up.
1. Pipette the 50 μL of the sample into a 1.5 mL
microcentrifuge tube.
2. Dilute the sample with 450 μL of D.I. water (10Â dilution) (see
Notes 14 and 15).
3. Transfer the diluted sample into the cuvette.
4. Insert the cuvette into the instrument (see Note 16).
3.4.2 Zeta Potential
Measurement
1. Pipette 75 μL of the sample into a microcentrifuge tube.
2. Dilute the sample with 675 μL of D.I. water (10Â dilution) (see
Notes 14 and 15).
3. Slowly inject the sample into the cuvette to prevent the introduction of air bubbles.
4. Insert the cuvette into the instrument (see Note 16).
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Yi-Hsuan Ou et al.
7 cells into 5 aliquots (200 μL/each).
5. Place each aliquot in a spin cup fitted with a 10 μm membrane
filter and centrifuge at 14,000 Â g, 4
C for 10 min. (see Notes
3 and 4).
6. Re-introduce the flow-through to the same spin cup and repeat
the centrifugation step (step 5).
7. Pipette the flow-through to another spin cup fitted with two
8 μm membrane filters and centrifuged 14,000 Â g for 10 min.
8. Re-introduce the flow-through to the same spin cup with 8 μm
membrane filters and centrifuge 14,000 Â g for 10 min.
9. Collect the flow-through of each spin cup (see Note 11).
3.3 Purification
Process
1. Pool the flow-through from five different spin cups to obtain
1 mL of the raw CDNs sample.
2. Equilibrate the Sephadex G-50 Size Exclusion Column (SEC)
with ice-cold PBS.
3. Add the raw CDNs onto SEC slowly to prevent disturbance of
the Sephadex G-50 beads.
4. Collect each 0.5 mL (one fraction) in a new 1.5 mL microcentrifuge tube. Fraction 3 and 4 are CDNs (see Note 12).
5. Pool the fractions (fraction 3 and 4) and store on ice temporarily for the characterization and further processings or at 4
C
for up to 3 days (see Note 13).
3.4 Characterization
3.4.1 Hydrodynamic Size
Measurement
The following protocol described the steps of using a Malvern
(Nano series) instruments. The protocol may need slight modifications for the application in other systems. The Zetasizer Nano
instrument should be switched on for at least 30 min before the
measurement to allow the laser to warm up.
1. Pipette the 50 μL of the sample into a 1.5 mL
microcentrifuge tube.
2. Dilute the sample with 450 μL of D.I. water (10Â dilution) (see
Notes 14 and 15).
3. Transfer the diluted sample into the cuvette.
4. Insert the cuvette into the instrument (see Note 16).
3.4.2 Zeta Potential
Measurement
1. Pipette 75 μL of the sample into a microcentrifuge tube.
2. Dilute the sample with 675 μL of D.I. water (10Â dilution) (see
Notes 14 and 15).
3. Slowly inject the sample into the cuvette to prevent the introduction of air bubbles.
4. Insert the cuvette into the instrument (see Note 16).
162
Yi-Hsuan Ou et al.