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specifically devised fluorescence-shielding membrane of similar material
(FATIMA plate). Alternatively it may be possible to use the conventional single-
well Transwell units which can be combined in the number desired by being
inserted into the wells of a conventional 24-well plate. Standard Unicell 24 and
conventional Transwells may be suited for migration analyses in which there is a
specific interest in monitoring the process by combined fluorescence and phase-
contrast microscopy, whereas FATIMA plates are specifically devised for
accurate and high-throughout put fluorescence-based assays. In all cases, the
choice of pore size of the membrane of the insert depends largely upon the cell
type and its relative size. For example, if neutrophils or smaller lymphocytes are
studied, inserts carrying 3-5-
m pore size membranes are recommended. For
anchorage-dependent cells it is preferable to use 8-12-
µ
m pore size membranes.
It is advisable to run some pilot assays to determine the type of membrane that
allows for the minimal “passive” transmigration rate, i.e., in the absence of mem-
brane-coating and/or chemoattracting agent.
µ
17.
Matrigel is currently the most commonly used 3-dimensional ECM substrate.
However, it should be emphasized that it is a poorly characterized murine sar-
coma-derived basement membrane ECM. If there is a specific need to work with
human material, the isolation of a human homolog to Matrigel,
Humatrix
(39)
,
has recently been described from smooth muscle cells. Interstitial-like ECMs of
desired compositions may readily be prepared by incorporating selected ECM
molecules during in vitro fibrillogenesis of interstitial collagens (mainly collagen
type I, III, and V; normally used at 0.5-1.5 mg/mL;
refs.
23
and
46
). Other pos-
sibilities to produce interstial ECM-like structures are through the generation of
fibrin clots from fibrinogen or the use of artificial biopolymeric matrices
(47)
.
Finally, if there is a specific interest in assaying native ECMs, several protocols
are described in the literature for the production of such matrices in vitro derived
from cultured cells or tissues.
18.
Some experimental protocols suggest that labeling of anchorage-dependent cells
while attached to plastic is ideal. This should result in an improved viability com-
pared to labeling in suspension after detachment of the cells from their growth
substrate. We have noticed, however, that for several anchorage-dependent cell
lines labeling in mobilized phase is less efficient than when done in suspension.
Therefore, we recommend to carry out cell labeling after detachment and by
diluting the fluorochrome in sucrose as indicated.
19.
In some cases it may be advantageous to label the cells on ice to allow the dye to
incorporate into the plasma membrane under reduced rate of endocytosis, thus
reducing the potential of dye accumulation in cytoplasmatic vesicles. Cell tag-
ging at this lower temperature requires a longer labeling time and may lead to a
compromising tagging. Leukocytes normally prefer a physiological labeling tem-
perature and a higher dye concentration. They also incorporate more efficiently
DiI-derivatives than other lipophilic dye variants, but, in some cases, may also be
refractory to these dyes. Labeling at 4
C may, however, be preferable when work-
ing with freshly isolated human leukocytes in which the tendency to self-aggre-
gate and become metabolically activated has to be prevented.
°
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