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nisms regulating cell migration, the binding force of a cell to the substratum
must be considered to be a critical parameter to establish. A number of recent
studies have centered upon this problem and have provided data on the relative
force of cell adhesion exhibited by diverse cell types onto cellular and ECM
substrates, both under static
(3-9)
and dynamic conditions (i.e., under shear
stress;
ref.
10
).
1.2. Cell Adhesion Assays Under “Static” Conditions
When it comes to cell adhesion under static conditions, a number of proce-
dures to quantify this process have been described in the literature and all of
them have capitalized on the use of mechanical forces to remove the nonbound
or weakly bound cells
(11)
. The methods by which these forces have been
applied to the bound cells include simple fluid flushing, the application of buoy-
ance
(12)
or rotating motions
(7
,
13)
, and centrifugation
(4-8
,
14
,
15)
. It was
early on proposed that, under static conditions, the application of a strictly
perpendicular removal force could yield a precise way to exert a definable and
measurable detachment force onto a population of cells
(4-8)
. Thus, although
considering all of the involved biophysical constrains and variables, in our
opinion this method still emerges as the preferred one. However, the centrifu-
gation assay procedure devised originally with the intent of quantitating weak
cell bindings, and the forces with which cells could bind to a given substratum
(16)
, had a number of drawbacks and limitations. We describe here a novel cell
adhesion assay which we have denoted Centrifugal Assay for Fluorescence-
based Cell Adhesion (CAFCA) (TECAN AG, Ropperswil, Switzerland) (
17
;
Fig. 1
), and which we regard to be the cell adhesion assay of choice for the
following reasons. The assay exploits the use of differential centrifugal forces
to achieve maximal accuracy and reproducibility, while maintaining the possi-
bility to precisely estimate the relative cell adhesion strengths. What we arbi-
trarily denote adhesion force (
A
fd
) can be calculated as dyn/cell, following the
generation of a suitable “force-dependent curve” to retrieve the force required
to detach 50% of the bound cells. The formula to adopt is then:
A
fd
= (
D
c
-
D
m
)
×
F
c
(where
D
c
is the specific cell density, intermediate value = 1.07 mg/cm
3
;
D
m
is the specific density of the medium, 1 mg/cm
3
;
V
c
is the volume of the
cell; and
F
c
is the centrifugal force yielding 50% cell detachment).
CAFCA is based on two centrifugation steps: the first one to allow for a
synchronized cell-substratum contact; and the second one (in the reverse direc-
tion) to allow for removal of the unbound/weakly bound cells under controlled
conditions (
Fig. 1
). The assay is unique in that it combines the possibility of
accurately estimating the cell-binding avidities while allowing a precise
assessment of the ratio of bound vs nonbound cells within a given cell popula-
tion. CAFCA is rapid (total assay time may be <1 h) and is applicable to a
V
c
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