From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 199

7.2. Controlling biological samples
Parameters such as temperature or pH can directly affect the chromophore
properties. In addition, these physical parameters plus other biologically rel-
evant parameters (osmolarity, ion and metabolite concentrations, etc.) may
alter the activity of signaling cascades (including kinases) and hence the mea-
surements obtained with KAR biosensors. Experimental conditions must
therefore be controlled in order to minimize the impact of such parameters.
7.2.1 Temperature
Concerning the temperature, experiments must be carried out at a stable
temperature (usually 37 C) inside thermostated space surrounding the
whole microscope. These incubators require hours to warm up and reach
a stable temperature and should always be turned on to ensure optimal sta-
bility. The temperature probe should be positioned as close as possible to the
sample. The thermal inertia due to contact with both the objective and the
microscope stage might need direct control with thermostatic controllers.
While temperature probes are most of the time positioned in air, there
can be a large difference between the average enclosure temperature and
the sample temperature. Calibration of this difference must thus be per-
formed by positioning the temperature probe in the medium. 144
7.2.2 Imaging media and perfusion
In long-term experiments, drying of the imaging mediummodifies ionic con-
centrations and pH. A buffered medium can be used for short-term experi-
ments (from 2-4 h depending on cell type). For longer measurements, the
use of a CO 2 incubator and a perfusion system to renew the culture medium
is recommended. In all cases, media containing phenol red should be avoided,
as this molecule is slightly fluorescent and leads to an unwanted fluorescent
signal. A phenol red-free medium must therefore be preferred. Details on
the preparation of dedicated imaging media can be found in Refs. 42,51 .
Perfusion systems are very useful to add drugs to the sample. Alterna-
tively, drugs in a preheated medium can be applied manually in the dish with
a Pasteur pipette. This can, however, lead to motion artifacts because of
shear stress, and some practice is necessary.
7.2.3 Biosensor expression level
Transfection agents or electroporation is often chosen to transfect cells with
biosensor plasmids. Viruses (e.g., adenoviruses or Sinbis) can be considered
for kinase activity measurement in cells resistant to the classic transfection