Biomedical Engineering Reference
In-Depth Information
can sometimes result in both brighter and more numerous
PLA spots, although too much antibody will lead to high
background fl uorescence. However, antibodies that are too
dilute will result in suboptimal assay performance. Choosing
an abundant target is also helpful. A viral epitope that is steri-
cally hindered or scarce may necessitate an increase in the
MOI to compensate for suboptimal signal strength.
4. In some cases, extending the amplifi cation time can improve
assay performance. It is best to begin with 2 h of incubation
and increase the amplifi cation time if weak signal intensity is
observed. We have used longer incubation time (up to 4 h) to
obtain stronger PLA signals for weak antibody pairs.
5. Although theoretically a nonproprietary mounting medium
such as ProLong
®
Gold Antifade Reagent with DAPI can be
used for this step, our experience with various mounting media
showed that the Duolink
®
version works well and consistently
for long-term preservation of PLA signal intensity.
6. The Duolink
®
ImageTool can be purchased from Sigma-
Aldrich, whereas BlobFinder can be downloaded free of charge
from the Centre of Image Analysis at Uppsala University
(
http://www.cb.uu.se/~amin/BlobFinder
)
. Both programs
are easy to learn and use and utilize similar methods for the
identifi cation of PLA signals. It is recommended that the selec-
tion of images for capture and analysis be done in an unbiased
manner using the DAPI channel. Also, cell populations to be
compared must be roughly similar in terms of the number of
nuclei as well as overall cell confl uency. Such an approach will
minimize variations in the average number of PLA spots per
cell and background fl uorescence.
It is essential to standardize the exposure time and threshold
values for all images within a particular channel to obtain an
accurate quantitative analysis. To reduce variability in signal
values among cells and improve overall statistical signifi cance,
at least 100 cells must be imaged and analyzed. Two methods
are available in BlobFinder to quantify signal levels: dot count
and fl uorescence intensity. In our experience, the dot count
option works particularly well for low signal intensity, while
the total fl uorescence intensity option is best for highly abun-
dant PLA spots.
7. Seed cells so that they are <50 % confl uent on the next day.
This will maximize effi ciency of infection and minimize differ-
ences in virus entry among the infected cells. At least several
hundred thousand cells must be plated to ensure the cell pellet
is visible upon harvest and to prevent cell loss during the
staining procedure.
As with any other technique, proper controls are crucial
for correct data interpretation. Two key negative controls
