Biology Reference
In-Depth Information
the assay. Thus, it is preferable to run the first centrifugation step at a lower force
than that used for cell-substratum adhesion, and additionally, this has to be
empirically set according to the cell type. In the case of lymphocytes binding to
the endothelium, for instance, we have observed that a force of 8-10
g
is an
adequate force to allow synchronized contact with the endothelial cell monolayer,
while minimizing the number of lymphocytes that are becoming constrained
between the single endothelial cells. The reversed centrifugal force may similarly
need to be differently adjusted when compared to that applied to cell-molecule
adhesion. Normally, a higher force is required to discriminate between true and
nonspecific cell binding. To ascertain that the parameter settings are optimal for
each individual experimental condition, it is advisable to observe the bound cells
under an inverted microscope, with or without the use of fluorescence.
14.
FATIMA (
Fluorescence-Assisted Transmigration Invasion and Motility Assay
)
is a versatile in vitro “cell migration assay” based on the transversing of tagged
cells through an inert porous micromembrane. The lather serves the sole purpose
to function as a physical barrier for differentiating “motile” versus “nonmotile
cells”; meaning cells that have exhibited the capability to actively locomote under
the influence, or in the absence, of a chemoattracting agent. According to our
definitions
Transmigration
(T
)
in the acronym refers to the process whereby a
cell is penetrating a single-cell monolayer (e.g., an endothelium or epithelium) or
a thin tissue section (<5-10
m in thickness).
Invasion
(I) refers to the process
whereby a cell penetrates and move through a multicellular structure, i.e., a
cellular multilayer, an explanted/ in vitro reconstituted tissue; a thicker tissue
section vibratome (<20
µ
m in thickness), or a 3-dimensional ECM.
Motility
(M
)
refers to the movement of a cell on a bidimensional ECM substrate.
µ
15.
Presently, there is a vast assortment of fluorochromes for intracellular labeling
(
see
the Molecular Probes' product catalog). We have recently carried out an
extended comparison between the presently available main categories of fluorescent
dyes having the capability to become taken up spontaneously by nonneuronal
cells (i.e., neurons may also be tagged by retrograde transport of single fluoro-
chromes or fluorescent microspheres through their axons/projections). These
include vital dyes based upon intracellular esterase activity; thiol-reactive fluo-
rescent compounds; and numerous lipophilic dyes. When carrying out long-term
migration assays, the otherwise very convenient vital dyes based on esterase
activity and thiol-reactivity are improper as they are completely released by the
migrating cells within less than 24 h. On the other hand, the lipophilic dyes,
which remain within cells for weeks, diffuse passively into the cells and may be
taken up to a significant extent also by dying cells. On the basis of this unavoid-
able trade-off concerning the fluorochrome choice, we find that lipophilic dyes
are the optimal cell labeling agents to use for long-term assays. The wide vari-
ety of lipophilic tracers currently available also provides a great flexibility as
well as the possibility to accomplish experiments involving multiple labeling of
two or more cell populations.
16.
Both Unicell 24 and FATIMA plates are provided in a 24-well format and carry
either a transparent, polycarbonate-based porous membrane (Unicell 24), or a
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