Biology Reference
In-Depth Information
CAFCA miniplates and those emitted by cells in the wells of the
top
CAFCA
miniplate), and thereby accurately determine the ratio bound vs nonbound cells
(
see below
). On the other hand, when working with small cells, such as lympho-
cytes and neutrophils, weak binding interactions are possible to detect by lower-
ing the centrifugal force down to ~10
g
.
11.
If there is a specific interest in knowing the exact number of cells bound to the
substratum and the number of cells that have failed to do so, a calibration curve
based on a serial dilution of equivalently labeled cells can be run in parallel to
extrapolate the corresponding cell numbers on the basis of the corresponding
relative fluorescence levels.
12.
For the achievement of optimal results, care should be taken to produce a cell
monolayer as homogeneous as possible, i.e., avoiding in as much as possible to
leave uncovered areas of the plastic. This would minimize the possibility for
nonspecific binding to areas of the well not covered by cells. It has to be taken in
consideration that in most cases, serum components that passively adsorb onto
uncoated plastic may promote cell adhesion (even when using fibronectin- and/
or vitronectin-depleted serum). Thus, it may generally be advantageous using
minimal concentrations of serum during the pregrowing of the “substratum” cell
monolayer. Moreover, if underlying cell monolayer can be produced by growing
the cells in serum-free medium, there is a possibility to carry out a “substrate
saturation” with a suitable blocking agent, similar to that described for cell adhesion
to ECM molecules (
step 4
). However, if this is not possible, it is advisable to
select a substrate molecule allowing the attachment of the cells selected to form
the underlying monolayer, but not adhesion of the cells to assayed for their capa-
bility to bind to this monolayer, even after a “conditioning” by serum-contained
factors (i.e., binding of these factors to the selected substrate molecule). For
instance, when we run the assay for examining the lymphocyte-endothelium
interaction, we find that we can pregrow most types of endothelial cells on a von
Willebrand factor substrate, whereas most lymphocytes fail to significantly
interact with this ECM molecule, independently of whether or not serum compo-
nents in the endothelial growth medium have bound to it or not. Alternatively, it
may be possible to pregrow the underlying monolayered cells in a “panning-like
fashion” onto wells precoated with a suitable antibody direct against a cell sur-
face-component specific for these cells. This provided that antibody ligation of
the targeted cell surface component does not cause significant changes in the
cell-cell adhesion behavior of the cells. It is also important to ascertain that cells
of the underlying cell monolayer do not detach from the substrate during the
reverse centrifugations. This is efficiently controlled for each single experimen-
tal situation by pretagging cells of the underlying cell monolayer with a red- or
blue-fluorescent CellTracker dye (Molecular Probes, Inc.) and then determining
the respective fluorescent signals in each well. Most microplate fluorometers,
including the SPECTRAFluor Plus, are equipped with several filter sets to allow
for multiple fluorescence detection.
13.
The procedure for cell-cell adhesion requires a more delicate parameter setting
because of the marked variability when two or more cell types are involved in
Search WWH ::
Custom Search