LABEL-FREE MONITORING OF CELL SIGNALLING PROCESSES THROUGH AFM-BASED FORCE MEASUREMENTS - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 352

minutes) and after (5 to 20 minutes) AngII stimulation. Indeed, in these time
intervals, the averaged luctuation increases from 0.70 ± 0.07 nm to 6.30 ±
0.46 nm in amplitude. A tenfold increase in the cell surface luctuations is
most likely indicative of the cell cytoskeleton remodelling. Monitoring of cell
response with the AFM requires the injection of signiicant volume (100-
500 μl) of buffer containing a cell receptor agonist (i.e. AngII) into the AFM
luid cell. This injection of luids could potentially inluence the cells and
thus the AFM signal monitoring through liquid low disturbance. Indeed,
a sharp spike is normally observed immediately after the injection of
500 μl of the buffer. However, this sharp discontinuity in the force signal
is not to be mistaken with a cellular response such as the one presented
in Fig. 17.2a . Figure 17.3 presents two control experiments conducted
to conirm the contribution of the AT 1 receptor stimulation in the height
response recorded with the AFM. As a control, HEK-293 cells transfected
with the AT 1 receptor were stimulated with HBSS (vehicle for all AngII
injection). As for the AngII stimulation experiment, the buffer HBSS is gently
introduced with a micropipette into the luid cell while the AFM signal is
recorded. As expected, no signiicant height increase of the cell body was
detected, nor any morphological changes observed. In an additional control,
the stimulation of MOCK HEK-293 cell (transfected with an empty vector)
with 100 nM AngII generated no contractile response, and no signiicant
difference is observed in the averaged signal luctuation before and after
(a)
(b)
Figure 17.3. (a) Control experiment in which HEK-293 cells, transfected with the
AT 1 receptor, are exposed to an injection of 500 μl of the buffer solution (HBSS)
alone. The AFM signal and the phase contrast micrographs at −2 and 10 minutes
conirm that the cells do not respond to such treatment. (b) Control experiment in
which HEK-293 cells transfected with an empty vector (without the AT 1 receptor
coding sequence) are exposed to AngII (in 500 μl HBSS). Injection spikes occur in
both experiments but no cell responses are seen. Reprinted with permission from
Ref. 23.