Biology Reference
In-Depth Information
7.
Assign for each set of assays a “blank.” For this purpose, utilize one of the inserts
in which both cells on top of the membrane and cells at its underside are wiped
off with a cotton swab and the fluorescence is read in the absence of cells.
8.
Calculate the percentage migrated/invaded cells by subtracting the “blank” from
the fluorescence values corresponding to the migrated cells.
3.2.4. Procedure for FATIMA Using
the Specifically Devised FATIMA Plates (
Fig. 4
)
1.
Steps 1-3
are identical to the procedure described in
Subheading 3.2.3.
2.
Measure the fluorescence from the top (corresponding to nonmigrated cells) and
bottom (corresponding to transmigrated cells) side of the plate (
see
Note 21
).
3.
Repeat the fluorescence measurement at different time-intervals (kinetic analy-
sis). TECAN AG, the supplier of the FATIMA system, provides a dedicated
software that automatically performs the calculations of the ratios transmigrated
cells/time unit (
see
Note 22
).
4. Notes
1.
The ideal coating concentration to use for each individual ECM molecule may
vary. This depends upon the cell type and the intrinsic capacity of the ECM mol-
ecule to become adsorbed onto plastic. We find that a coating concentration range
of 0.01-10
g/mL is suitable for a wide range of ECM proteins when carrying
out dose-dependency tests of cell-substratum attachment. In our experience, a
coating concentration of 0.01
µ
g/mL can be adopted as a starting concentration
when designing a coating concentration curve. A lower concentration usually
yields in sufficient amounts of the ECM molecule (we have tested >30 different
ones) becoming bound to plastic. If there is a specific interest in preparing a cell-
adhesion substratum containing a mixture of ECM molecules, the total protein
concentration in the coating solution may obviously be raised to assure that a
sufficient amount of the less represented molecules in the mixture is obtained.
However, caution should be taken when coating with single or mixtures of ECM
components that have an intrinsic propensity to self-assemble spontaneously (or
form heterogenous assemblies), even in the absence of divalent cations, physi-
ological pH and temperature, or other assembly-promoting factors. Examples of
ECM components with this tendency are fibronectin, vitronectin, von Willebrand
factor, laminins, and collagens. The precise amount of protein bound to the spe-
cific PVC-based CAFCA miniplates has been determined for a number of ECM
components
(5
,
6
,
17)
.
µ
2.
We find that protein coating at 4
C overnight in the indicate bicarbonate buffer
yields the optimal adsorbance of the molecules onto plastic both in terms of
amount and “active” configuration of the immobilized molecule. It also largely
prevents unwanted multimeric complex formation of the molecules.
°
3.
A 1% solution of denatured BSA is a suitable blocking agent, i.e., its acts well in
saturating areas of the plastic left uncoated by the ECM molecule (this is espe-
cially important when carrying out accurate dose-dependency tests), and it is pre-
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