Biology Reference
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cells from the upperside of the membrane (
Fig. 3
). Although the sensitivity of
this method is high, it does not resolve a fundamental problem of the proce-
dure, namely to be able to precisely quantify the relative ratio transmigrated vs
nontransmigrated cells within the same population, and to determine the kinet-
ics with which the transmigration occurs. It should be also noted that there is
an intrinsic caveat in using fluorescent dyes with relative low intracellular
retention time when performing long-term migration assays, in that the sensi-
tivity of the assay invariably declines with time.
We describe here an improved fluorimetric assay that has the potential to
circumvent the above problems and has been denominated fluorescence-
assisted transmigration invasion and motility assay (FATIMA, TECAN AG).
The system may utilize conventional single unit Transwells, Unicell 24 plates
(
Fig. 3
) or a newly devised variant of these latter plates denoted FATIMA
plates (Whatman/Polyfitronics, Boston, MA) (
Fig. 4
). The assay may be
performed according to two different protocols depending on which of these
units is employed (
Figs. 3
and
4
). The unique trait of the FATIMA plates is
the incorporation of a specific porous membrane, which shares most of the
properties of the conventional polycarbonate membranes, but is devised such
as to shield fluorescent light in the wavelength range 450-550 nm. FATIMA is
based on the indiscriminate cell labeling with lipophilic carbocyanines dyes
(e.g., DiI, DiO, DiA, DiR and derivatives, Molecular Probes, Inc.), or the more
specific molecular tagging through GFP-based vectors.
Lipophilic fluorescent dyes are particularly suitable for long-term labeling
and tracking of cells in vivo and in vitro
(43-45)
and are therefore the pre-
ferred fluorochromes when cell migration assays are run under extended peri-
ods of time. Additional advantages of these fluorochromes include their
minimal spontaneous release from cells and the limited increase in the global
fluorescence intensity obtained upon cell division (when using isotope-tagged
cells this parameter has to be taken into account). When working with
Fig. 3.
(opposite page)
Schematic representation of the FATIMA procedure for
anchorage-dependent
(left)
and suspension-growing
(right)
cells, based on the use of
conventional Transwells or plates that carry transparent membranes. Fluorescence
detections
(blue boxes)
are performed at three different times: initially to determine the
total number of cells aliquoted into the Transwells; after removal of the nontransmigrated
cells; and at the end of the experiment after removal of all cells from the Transwells.
These latter fluorescence measurements serve to determine the background fluores-
cence, i.e., including that deriving from any possible dye release from the cells during
migration and the fluorescence that may remain associated with the substrate and/or
polycarbonate membrane. When working with anchorage-dependent cells, complete
removal of “nontransmigrated cells” is an absolute requirement for obtaining reliable
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