Fluorescence Assays to Study Cell Adhesion and Migration In Vitro - Methods in Molecular Biology: Extracellular Matrix Protocols - page 319

concentrations. For optimal fluorescent measurements purposes ( Fig. 1 ), India
ink (at the optimal concentration of 2%) turns out to be the most effective, inert
fluorescence quencher. We have ascertained for a number of cell types that incu-
bation in medium containing this concentration of India ink does not affect the
proliferation rate or cell adhesion behavior of cells.

7. The number of cells seeded per well may vary depending upon the size of the

specific cell type. We find that a concentration of 30,000 cells/well is ideal for
suspension-growing cells, whereas 1000-5000 cells/well are optimal when
examining larger anchorage-dependent cells. The minimal amounts of cells per
well that can be used in CAFCA are ≤ 1,000/well and ≤ 100/well for lymphocytes

and fibroblastic cells, respectively.

8. This is one of the first critical steps of the procedure. First, we find that a force of

142 g is an ideal centrifugation force to bring all cells (both lymphocytic and
fibroblastic) contained by each well in simultaneous contact with the substratum.
Moreover, we find that the centrifuges sold by Juan (France) are the most conve-
nient ones since they are precise and can accommodate 4 × 4 CAFCA miniplates

(i.e., 4 96-well plates) per centrifugation. The length of the subsequent incuba-

tion at 37

×

C may be varied as desired, although 15-20 min is the time-period that
we find necessary to allow for a stable cell adhesion to ensue.
If there is a specific interest in analyzing the receptor-ligand interaction with-
out involvement of the cytoskeleton or signal transduction phenomena, we find
that the entire procedure of CAFCA may effectively be accomplished at 4

°

C, as
shown for its predecessing adhesion assay (4 , 5 , 8) . In such a case, all the solutions
should be precooled to 4

°

C in a cooled centri-
fuge. Here, there is no need to perform an incubation of the cells after the cen-
trifugation step, but if there is a specific reason to do so, it obviously should be
carried out at 4

° C and the centrifugations run at 4 °

° C.

9. This is an extremely important step that may determine the final outcome of the

experiment. Care has to be taken to adequately fill the wells of the bottom and
top CAFCA miniplates such as to avoid air-bubble formation during the subse-
quent assembly of the plates. This means that wells of both plates have to con-
tain an excess of liquid (forming a bulging meniscous), such as to assure that
no air is trapped between the upper surfaces of the wells during face-to-face
assembly ( Fig. 1 ).

10. Differential centrifugation forces to detach the “weakly bound” or nonbound cells

are used to determine the relative binding avidity of the cells to the substratum. It
should be noted that firmly bound cells, i.e., those binding with high avidity may
not be removed with forces below those ascertained to retain viable cells (we
have determined the threshold to be a force of 750 g ). Thus, when a “force-

dependent” centrifugation curve is generated, a suitable force range to adopt is
~10-~750 g . We have observed that forces <40 g may not be sufficient to displace
the nonbound fibroblastic cells to the top CAFCA miniplate wells: this is an
absolute requirement for being able to detect physically separated fluorescence
signals (i.e., those emitted by substrate-bound cells in the wells of the bottom

≤