Biology Reference
In-Depth Information
important. A common and widely used procedure to block uncoated regions
of the substrate is to use bovine serum albumin (BSA). The substrate with the
desired ligand is incubated with 1% w/v BSA for ~1 hour at room temperature
just before measurements. To block hydrophilic surfaces, such as nontreated
polystyrene, 1% Pluronic F108 (BASF), a nontoxic triblock copolymer, has
also been used, providing negligible unspeciic adhesion. In addition, cells are
compliant bodies. Thus, a small compression force will lead to a relatively big
area of contact between the cell and the substrate, increasing proportionally
the probability of bond formation.
39
For example, a cell of ~5 μm radius and
1 kPa Young's modulus compressed against a lat surface with a force of 50
pN would provide an area of contact of ~1 μm
2
, assuming Hertzian elastic
contact.
39
On such a relatively big contact area, it is dificult to form individual
bonds. For this reason, it is sometimes necessary to reduce the ligand
concentration to very low levels and always work at minimal compression
forces. Another strategy to reduce the area of contact between the cell and
the substrate is to increase the approaching speed. Given the viscoelastic
nature of cells, the faster the approach velocity, the stiffer the cell will appear.
Thus, at a same compression force, increasing the approach velocity can help
to reduce the area of contact. Moreover, faster approach will provide shorter
contact times, reducing also the probability of bond formation.
A common property of living cells, particularly blood cells, is their ability
to form membrane tethers. These membrane nanotubes are formed when
a molecule on the cell surface being pulled is released from the underlying
cytoskeleton and thus membrane is allowed to low following the pulled
molecule.
When pulled at constant speed, the tether exerts a constant,
friction force against the cantilever tip
(
Fig. 11.4d
). As we will see in the next
section, in DFS measurements, in which complexes are stretch at constant
loading rates (force-time), the formation of tethers is not the optimal
condition. However, we can take advantage of this property of cells to carry
out alternative measurements.
When probing adhesion with AFM on adherent cells, such as ibroblasts
or endothelial cells, we can use cell culture dishes or coverslips on which
adherent cells are commonly grown
(
Fig. 11.4c
)
. Suspended cells can also
be immobilized on the substrate using similar approaches as the ones
described before for immobilization on AFM cantilevers. The advantage of
using this coniguration on adherent cells is that there is no need to detach
them from the substrate of culture, a procedure that may stress the cell.
With the cell immobilized on the substrate, the AFM tip has to be coated
with the biomolecule of interest. In that case, the tip geometry will deine
the area of contact. The commonly used pyramidal tips have the advantage
40,41
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