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used a colloidal probe coated with
vitronectin (VN) molecules to map the location of VN receptors on the
surface of live osteoblastic cells
In their next work, Kim
et al.
and of prostaglandin receptors on living
CHO cells 43 (details will be given later on). Since the method was based on the
interactions between a fairly large number of ligand molecules on a colloidal
probe and a corresponding number of receptors on the cell surface, a coarse
grained map was available over a large area and the result was useful to
make correspondence with that of the conventional labelling method using
luorescent antibodies and to map receptors on the live cell surface with high
conidence. Previously available methods based on luorescence labelling
relied on ixing of the cell, which is fatal. Instead of counting the number of
receptor molecules on the cell surface, Kim
42
et al.
used the separation work as
deined earlier by Eq. (12.1).
With this method, the distribution of VN receptors on a living murine
osteoblastic cell was successfully measured. First, the distribution of the
integrin α V β 5 subunit was conirmed by conventional immunohistochemistry
after ixing the cell. To visualize the distribution of the receptor on a living cell
by an independent and potentially a more quantitative method, the AFM was
used with a micro-bead attached to the cantilever end to increase the area of
contact and VN was immobilized on the micro-bead, and from the resulting
force curve, separation work was calculated and displayed as described
earlier.
Kim
42
et al.
further
studied the distribution of EP3 receptors on a living CHO
cell.
Green luorescent protein (GFP) was fused to the extracellular region
of the EP3 receptor on a CHO cell and a micro-bead was coated with anti-
GFP antibody. The interactions between the antibodies and GFP molecules
on the cell surface were recorded, and the result indicated that EP3 receptors
were distributed on the CHO cell surface not uniformly but in small patches
coincident with the result of immuno-histochemical observations. Repeated
measurements on the same area of the cell surface gave conirmation that it
was unlikely that the receptors were extracted from the cell membrane during
the experiments. The separation work required to break a single molecular
pair was estimated to be about 1.5 × 10
43
J. Using this value, the number of
EP3 receptor on the CHO cell surface was estimated to be about 1 × 10
18
4
under
the assumption that the area of the cell surface was about 5000 μm
.
The colloidal probe method can cover a wide area of approximately 20
μm ×
2
20 μm. Much wider areas may be covered by systematically shifting
the area for scanning or by using colloidal probes of larger diameter, but as
the number of ligand receptor interactions pairs increases, it will be harder
to dissociate the probe from the cell surface, making it impossible to map
receptor molecules.
 
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