Biology Reference
In-Depth Information
Application of FRET and Microscopy
2.
Change the medium of the cells after 3-4 h from DMEM (FCS) to DMEM
(FCS/PS).
3.
Let the cells grow at 37
C/5% CO
2
overnight. Overnight transfection is
sufficient for proper expression of plasmids. The cells can be used for spectral
and FRET measurements at any time, usually 14-24 h after transfection.
14.3.2
Cell lysis for Fluorolog
1.
Keep the cells on ice.
2.
Remove the media and wash the cells with 700-1000
m
l of PBS.
3.
Mix buffer A (200
m
l/plate) and two inhibitors (10
m
l/ml) (PMSF100 mM and
).
4.
Add the inhibitors to cells and keep at 4
C for 15-20 min with shaking.
5.
Centrifuge the cells for 15 min at 14,000 rpm at 4
C.
6.
Remove the pellet carefully and transfer the supernatant to a cuvette and bring the
volume to 2 ml by adding buffer B and further proceed with experiment (like
titration measurements over time).
7.
For spectral analysis measurements, lysing of cells is not required. Bring the cells
from incubator and remove the media and wash the cells with 1 ml of PBS
once. Then, detach the cells from the plate by adding 1 ml of buffer B twice by
gently sucking in and out with 1 ml pipette so that the cells are alive for the
experiment. Transfer 2 ml volume of cells to cuvette and continue with
experiment.
CLAP100
14.3.3
Procedure part I: data acquisition
Quantitative lux-FRET measurement requires acquisition procedure shown in the
flowchart (
Fig. 14.3
). For the spectral separation of donor and acceptor fluorescence
contribution, reference spectra of cells transfected with only donor and only acceptor
and others must be obtained for each excitation wavelength under identical acquisi-
tion settings (i.e., excitation intensity, exposure time, and detector gain) as in the
FRET experiment. From that reference measurements,
F
i
,ref
(l) and
F
i
,ref
(l) are
obtained. In case of multicolor experiments, all other spectral components must
be obtained as separate measurements in a similar manner. Especially for experi-
ments with a fluorescence spectrometer, measuring the autofluorescence and the wa-
ter Raman spectrum is required. Accordingly, Eq.
(14.2)
must be extended for
spectral unmixing. Data acquisition of a standard tandem construct is required to de-
termine
R
TC
. Dependent on the experiment, positive and negative control must be
also recorded. The real FRET experiment with the sample of interest should then
be recorded, best under identical conditions. Take into account that all reference
measurements are required for later data evaluation. Thus, it is advisable to prepare
some additional reference samples as well as to acquire references and controls be-
fore starting extensive sample experiments.
