From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 195

PhosphoTag western blotting. The overall amounts of expressed biosensors
are then revealed by an anti-GFP antibody. 127
6.6.2 In cellulo characterization
In cellulo characterization consists of expressing the biosensor in a mammalian
cell model and assessing the ratiometric signal in the presence of specific ac-
tivators and inhibitors of the kinase of interest. Biosensor control constructs
(earlier version or/and non-phosphorylatable biosensor) should undergo
similar experimental conditions. Transient transfection of plasmid DNA
encoding biosensors is an accessible and inexpensive approach, which is
sufficient to produce biosensors using cellular transcriptional machinery.
Kinase activity measurement in a cellular context can also evaluate the spec-
ificity of a biosensor candidate: kinase blockers should prevent the rati-
ometric response to activators, demonstrating the involvement of one
specific kinase family in the process being studied. This strategy is more often
seen as a complementary rather than an alternative method to evaluate
FRET efficiency of candidate sensors.
In vitro and in cellulo characterizations are often used to evaluate quanti-
tatively the effect of several kinase inhibitors to determine the most effective
inhibitor of the kinase of interest. Indeed, they can serve as high-throughput
screening to evaluate rapidly the effects of several pharmacological treat-
ments on the activation or extinction of a particular pathway. For such as-
says, it is essential that the biosensor exhibits a robust and reproducible signal.
This is generally assessed by the Z -factor, which is a statistical parameter that
compares the dynamic range to data variation. 123 A schematic representation
of such an experimental design is shown in Fig. 5.23 .
7. CONSIDERATIONS FOR KAR MEASUREMENTS
7.1. Controlling acquisition systems
As previously described, several techniques allow quantifying FRET for
KAR imaging. Each of them requires a dedicated setup and thus the
corresponding characterization procedures. The most advanced acquisition
techniques (spectral acquisition, TD and FD FLIM, etc.) mainly require
rigorous characterization of the systems and optimized acquisition condi-
tions as described in the literature. 23,26,59,139 They can then be directly
used for KAR imaging and yield unambiguous quantification of FRET
efficiencies. Ratiometric imaging, despite being an easier acquisition
procedure, can results in erroneous interpretation of FRET efficiency due
to the classical drawbacks of quantitative fluorescence imaging. The