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24. Draw a gate identifying CFSE low cells and other for the CFSE high
cells.
25. Collect 5,000-10,000 total CFSE positive cells.
26. Determine the percentage of CFSE high and CFSE low cells.
27. Calculate the ratio of unpulsed to pulsed target cells in the
naïve mouse. This will be defi ned the 0 % lysis level.
28. The percent of specifi c lysis is determined by loss of the antigen-
pulsed CFSE low population compared with the non-pulsed
CFSE high control population using the next formula:
(
)
1
ratio in na ve mouse
/
ratio in experimental mouse
×
100
.
4
Notes
1. There are several commercially available kits for Ad titration
based on anti-hexon staining (e.g., Clontech X-rapid Titer;
Agilent Ad-Easy Viral Titer, Cell Biolabs Quick Titer, etc.).
2. During this time cells are infected, attach, and form a mono-
layer. The time to achieve a maximum accumulation of virus
protein will be longer when tittering in less permissive cells
lines or for viruses with lower replication effi cacy. If these
parameters are unknown, a 48 h incubation can be tried.
The differential titer of one virus in two different cell lines is a
parameter that indicates the permissiveness of the cells to that
particular virus.
3. Count groups of adjacent green cells as a single infectious unit
as these adjacent cells derive from a single infectious unit.
4. If plaque assay is done in HEK293 cells or a cell line that does
not attach well to the plate this step may be skipped to main-
tain the monolayer.
5. Alternatively, stain with Thiazolyl Blue Tetrazolium Bromid
(MTT) adding 0.1 mL/well of a 5 mg/mL MTT solution pre-
pared in PBS or DMEM 5 % FBS, and incubating in darkness
at 37 °C and 5 % CO 2 for 3 h.
6. This assay is simplifi ed if a vector with a reporter gene is used
(e.g., GFP, luciferase) as it offers a direct read out of the amount
of reporter virus (FACS, fl uorescence, or luminescence).
7. A549 cells are also a good substrate cell line to detect Nab, and
they attach better than HEK293 cells to the plastic. If these
cells are used we recommend using and MOI of 2.5 iu/cell for
the assay instead of 0.25 iu/cell. For standardization and com-
parison between assays a reference neutralizing polyclonal anti-
body (e.g., ab6982, Abcam) should be used as a test control
antiserum.
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