Biology Reference
In-Depth Information
7. Incubate at 37 °C 5 % CO 2 in incubator and observe daily
under the microscope the appearance of CPE. Leave until par-
tial CPE can is seen in the sixth or seventh column (5-8 days
post-infection). First column should show complete CPE.
8. Prepare BCA protein stain.
9. Shake softly the 96-well plate in order to resuspend detached
cells and aspirate the medium from every well with care. Wash
gently cells with 200
L of PBS to eliminate the remaining
FBS that would mask the differences in protein content.
10. Remove the PBS and quickly add 200
μ
μ
L/well of BCA protein
stain with a multichannel pipette.
11. Leave 30 min at 37 °C and read absorbance in spectrophotom-
eter at 540 nm. Use as blank any well from fi rst column.
12. Record results in an excel fi le and plot the amount of protein
as a percentage of the noninfected control against vp/mL or
iu/mL.
13. Calculate the IC50 for each virus by nonlinear regression using
an adapted Hill equation (e.g., GraFit software, Erithacus,
Horley, UK).
1. Seed HEK293, A549, or the desired cell line in 6-well plates in
order to have 90 % confl uence in the time of infection. Before
the infection, remove the medium and add 0.9 mL of DMEM
5 % FBS to each well.
2. Prepare 1/10 dilutions of the virus stock or cell extract (CE)
and add 100
3.3 Plaque
Size Assay
L of each dilution per well. Proceed from the
most diluted to the most concentrated in order to use the same
pipette tip.
3. Incubate at 37 °C/5 % CO 2 for 4 h.
4. Prepare autoclaved 1 % agarose in H 2 O and temperate to 50 °C
(previously prepared agarose can be melted in a microwave).
5. 4 h after infection, remove infection medium and wash cells
with PBS ( see Note 4 ). Remove medium and PBS starting
from the diluted wells to prevent contamination. Handle one
plate at a time.
6. Mix 7.5 mL of 1 % agarose (at 50 °C) and 7.5 mL of DMEM
5 % FBS (at 37 °C) in a falcon tube. Remove the PBS and add
2 mL of the agarose-medium mix in each well. Leave 10 min
on the hood without lid. Repeat this overlay for each plate.
7. Add 2 mL of DMEM 5 % FBS to each well and transfer to the
cell incubator.
8. Incubate at 37 °C and 5 % CO 2 until plaques appear. If the
medium acidifi es (turns yellow) replace it carefully.
9. Stain plaques with neutral red adding 1 mL of 0.03 % neutral
red into each well and incubating O/N at 37 °C and 5 % CO 2 .
μ
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