Biomedical Engineering Reference
In-Depth Information
It is critical to include positive and negative controls with
every PLA experiment, although the exact number and nature
of controls will depend on the specifi c details of each experi-
ment. Examples of essential negative controls include unin-
fected cells as well as infected cells processed for PLA in the
absence of one or both primary antibodies. Another useful
negative control may involve assaying for proximity between
two proteins that are normally localized to distinct subcellular
compartments. The best positive control is one that measures
interaction between an abundant pair of proteins—one viral
and one cellular—that are known to associate during infection.
If such a positive control is unavailable, two interacting cellular
proteins can be targeted instead. The measurement of proxim-
ity between two subunits of a protein complex may be a good
internal control for testing optimal reagent performance over
the time. Repeated assays of two proteins that demonstrate
large fl uctuations in signal intensity presumably indicate an
issue with either a reagent or experimental technique.
Our experience with the proximity ligation assay has
revealed a number of additional parameters that may affect
data quality and reproducibility. One factor is cell confl uency
prior to infection. Highly confl uent cultures generally exhibit a
lower average number of PLA spots per cell compared to cells
plated at low density. This appears to be a consequence of cell
crowding and thus a decrease in cell size. Another factor is
background fl uorescence. Typically, the staining of cells with
PLA reagents in the absence of primary antibodies results in
only a few nonspecifi c signals per cell. If more than several
spots per cell are observed for the negative control, it may indi-
cate a need for further troubleshooting of either experimental
conditions or assay implementation.
Because proximity ligation assays to measure virus co-
localization with cellular structures rely on both host and
microbial proteins, proper infection conditions are essential for
robust assay performance. A number of experimental parame-
ters can be adjusted to optimize signal strength and/or speci-
fi city. Conditions that can be adjusted include, but are not
limited to, the specifi c virus strain and host cell used, viral mul-
tiplicity of infection (MOI), synchronization of viral entry by
preincubation at 4 °C followed by shift to permissive tempera-
ture such as 37 °C, and selection of appropriate time points
during infection.
2. It is recommended that fi xation be done via gradual addition of
the cold fi xative directly into the cell culture medium. Such an
approach minimizes cell stress and subsequent cell detachment
from the coverslip surface during aspirations and washes. For
convenience, the fi xation, permeabilization, and blocking steps
can be done directly in the tissue culture plate containing the
