(e) Step 5: Incubate the plasmid–calcium phosphate complex
at room temperature for 20 min with no movement.
(f) Step 6: Gently drop the plasmid-calcium phosphate complex solution onto the surface of the media (see Note 23)
and gently rock the cell culture dish 2–3 times.
5. Incubate the transfected and control cells under 5% CO 2 at
37
C for 3 days.
3.3 Zika VLP
Collection
and Purification by
Discontinuous Sucrose
Gradients
1. Collect and pool (see Note 22) the Zika VLP-enriched OptiMEM
® media (~40 mL) into a 50 mL centrifuge tube and
centrifuge at 1000 Â g for 5 min to remove floating cells.
Collect the media from the mock transfection and treat in
parallel.
2. Filter the Zika VLP-enriched media through a 0.22 μm filter
(see Note 24).
3. Transfer the Zika VLP-enriched media into a 100 kDa cut off
column and centrifuge at 5000 Â g for 10 min at 4
C to
concentrate the Zika VLP. The final volume will be about
1.5 mL of concentrated Zika VLP in Opti-MEM
® media.
Reserve 200 μL for subsequent western blot analysis (Subheading 3.4).
4. Add 4 mL of 60% sucrose into an Ultra-Clear Tube.
5. Gently overlay 4 mL of 30% sucrose onto the 60% sucrose in
the Ultra-Clear Tube (Avoid disruption of the surface between
the two layers) (see Note 25).
6. Mix 1.3 mL of concentrated Zika VLP in Opti-MEM
® media
with 2 mL of 10% sucrose then gently overlay onto the two
layers of 30% and 60% sucrose in the Ultra-Clear Tube, again
avoiding disruption between the surface layers.
7. Gently put the Ultra-Clear Tube containing the three layers
into an ultracentrifugation bucket and fill with 10% sucrose
onto the upper layer until full and close the lid (see Note 26).
8. Perform the centrifugation at 134,434 Â g for 2 h at 4
C with
no brake (see Note 27).
9. Collect the visible band (concentrated Zika VLP) between the
30% and 60% sucrose layers by side puncture and put into a new
Ultra-Clear Tube (Fig. 2).
10. Add TNE buffer into the collected visible band in the UltraClear Tube until full then mix by inverting the tube and
centrifuge at 134,434 Â g for 1 h at 4
C with brake.
11. Discard the TNE buffer by pouring from the tube until no
excess liquid is left, the Zika VLP can be observed as a white
opaque pellet. Then add 50 μL of new TNE buffer onto the
Zika Virus-Like Particles
197
at room temperature for 20 min with no movement.
(f) Step 6: Gently drop the plasmid-calcium phosphate complex solution onto the surface of the media (see Note 23)
and gently rock the cell culture dish 2–3 times.
5. Incubate the transfected and control cells under 5% CO 2 at
37
C for 3 days.
3.3 Zika VLP
Collection
and Purification by
Discontinuous Sucrose
Gradients
1. Collect and pool (see Note 22) the Zika VLP-enriched OptiMEM
® media (~40 mL) into a 50 mL centrifuge tube and
centrifuge at 1000 Â g for 5 min to remove floating cells.
Collect the media from the mock transfection and treat in
parallel.
2. Filter the Zika VLP-enriched media through a 0.22 μm filter
(see Note 24).
3. Transfer the Zika VLP-enriched media into a 100 kDa cut off
column and centrifuge at 5000 Â g for 10 min at 4
C to
concentrate the Zika VLP. The final volume will be about
1.5 mL of concentrated Zika VLP in Opti-MEM
® media.
Reserve 200 μL for subsequent western blot analysis (Subheading 3.4).
4. Add 4 mL of 60% sucrose into an Ultra-Clear Tube.
5. Gently overlay 4 mL of 30% sucrose onto the 60% sucrose in
the Ultra-Clear Tube (Avoid disruption of the surface between
the two layers) (see Note 25).
6. Mix 1.3 mL of concentrated Zika VLP in Opti-MEM
® media
with 2 mL of 10% sucrose then gently overlay onto the two
layers of 30% and 60% sucrose in the Ultra-Clear Tube, again
avoiding disruption between the surface layers.
7. Gently put the Ultra-Clear Tube containing the three layers
into an ultracentrifugation bucket and fill with 10% sucrose
onto the upper layer until full and close the lid (see Note 26).
8. Perform the centrifugation at 134,434 Â g for 2 h at 4
C with
no brake (see Note 27).
9. Collect the visible band (concentrated Zika VLP) between the
30% and 60% sucrose layers by side puncture and put into a new
Ultra-Clear Tube (Fig. 2).
10. Add TNE buffer into the collected visible band in the UltraClear Tube until full then mix by inverting the tube and
centrifuge at 134,434 Â g for 1 h at 4
C with brake.
11. Discard the TNE buffer by pouring from the tube until no
excess liquid is left, the Zika VLP can be observed as a white
opaque pellet. Then add 50 μL of new TNE buffer onto the
Zika Virus-Like Particles
197