Biology Reference
In-Depth Information
FATIMA, a suitable computer-interfaced microplate fluorometer with
independent top- and bottom-side measurement capabilities, such the
SPECTRAFluor Plus (TECAN AG), should be employed to determine the levels
of corresponding “top/bottom” fluorescent signals of the plate. FATIMA fur-
ther has the advantage that it takes into account also “spatial” parameters.
Especially when analyzing anchorage-dependent cells, we have noticed that
transmigrated cells tendentially accumulate at the perimeter of the membrane.
Thus, by utilizing a microplate fluorometer equipped with the capability of an
automated multiple point-measurement mode to detect fluorescence in differ-
ent areas of the well surface (e.g., the SPECTRAFluor Plus is capable of mea-
suring 25 different points in a well of a 24-well plate) there is the possibility of
rapidly quantifying the spatial distribution of the migrated cells. This possibility
can further be exploited when migratory substrates with a spatially heterogenous
configuration are investigated. Apart from the possibility of accomplishing
kinetic studies of cell movement, there is an unparalleled capability of
FATIMA; this is the possibility of determining within few minutes and simul-
taneously the precise ratio transmigrated cells in up to 24 inserts.
2. Materials
2.1. CAFCA
1. Cells to be assayed for their binding capability to molecular substrates and cells
to be assayed in a monolayer arrangement for their capability to bind to other cells.
2. 0.05
M
bicarbonate buffer, pH 9.6: prepare a NaHCO
3
solution of 4.2 g/L and
one solution of Na
2
CO
3
of 5.3 g/L and mix the two components to reach the
desired pH; this takes less Na
2
CO
3
than NaHCO
3
.
3. ECM component(s) to test as substratum.
4. Bovine serum albumin (BSA, fraction V).
5. PBS: 8 g NaCl, 0.2 g KCl, 1.44 g Na
2
HPO
4
· H
2
O, 0.2 g KH
2
PO
4
in 1 L apyrogen
H
2
O.
6. PBS containing 5 m
M
EDTA (0.84 g/L Na
2
-EDTA in PBS; to be used only for
anchorage-dependent cells).
7.
Culture medium, RPMI (for suspension-growing cells) and DMEM (for anchor-
age-dependent cell) both supplemented with 15% FCS.
8.
Vital fluorochrome calcein AM (dissolved in DMSO at 2 mg/mL as stock solu-
tion; Molecular Probes, Inc.).
9.
Serum-free culture medium containing 0.1-0.5% polyvinylpyrrolidone (
M
r
360,000; PVP).
10.
Serum-free medium containing 0.1-0.5% PVP and with 2% v/v India ink.
11.
CAFCA miniplates (TECAN Group, Austria;
Figs. 1
and
2
).
12.
Semihydraulic vice (TECAN Group;
Fig. 2
).
13.
CAFCA holders (TECAN Group;
Figs. 1
and
2
).
14.
Assembly devices (TECAN Group;
Fig. 2
).
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