Biology Reference
In-Depth Information
tact the two liquid meniscuses and then press the
top
CAFCA miniplate onto the
bottom
one allowing the former to attach firmly to the double-sided tape of the
bottom
plate. At this point an “air-bubble-free” communicating chamber should
have formed (
Fig. 1
). Place the second light-metal device (
Fig. 2
) over the top
CAFCA miniplate and place the entire unit under the vise (
Fig. 2
) to tighten the
affixed plates.
11.
Remove the light-metal devices and place the assembled CAFCA miniplate unit
into the assembled black CAFCA hard-plastic holders (
Fig. 2
) and tighten the
holders. These units can now be reversed centrifuged for 5 min at the desired
centrifugal force (standard force = 46
g
;
Fig. 1
) (
see
Note 10
).
12.
Measure the fluorescence signal emitted by cells in wells of the top (
nonbound
cells
) and bottom (
substrate-bound cells
) sides of the CAFCA miniplates inde-
pendently (
Fig. 1
), ideally using a microplate fluorometer such as the
SPECTRAFluor Plus capable of detecting the fluorescence emanated from both
the top and bottom side of the microplate. The percentage bound cells, out of the
total amount of cells introduced into the system, can be calculated as: bottom
fluorescence value/bottom fluorescence + top fluorescence values. If the above
indicated computer-interfaced instrument is used, a dedicated CAFCA software
is provided by the supplier that automatically runs the calculations (
see
Note 11
).
3.1.2. Procedure for Cell-Cell Adhesion Assay
1.
Seed the cells to be used as the underlying (substrate) cell monolayer into the
CAFCA miniplate wells (make to use the presterilized type) at a concentration
earlier estimated to yield confluency by the following day, or whenever the
experiment is intended to be performed (
see
Note 12
).
2.
Once it is time to run the experiment, the procedure is the same as described for
cell-substratum adhesion, i.e.,
steps 5-12
(
see
Note 13
).
3.2. FATIMA
3.2.1. Preparation of the Membranes
1.
Cell motility:
Coat the membrane of the FATIMA plate, Unicell 24, or Transwell
(Costar) as indicated for CAFCA, considering that polycarbonate may adsorb
protein 10-50-fold less efficiently than the PVC plastic. The amount of coating
solution should be sufficient to cover the entire membrane surface. We recom-
mend to use a 100-
L volume for the individual units of FATIMA plates, Unicell
24 plates, and Transwells with a 6.5-mm insert diameter (0.33 cm
2
area; corre-
sponding to those fitting a conventional 24-well plate), and a 250-
µ
L volume for
Transwells with a 12-mm insert diameter (1.0 cm
2
area). These correspond to the
only commercially available units carrying membranes with 12-
µ
m pore size and
fitting a 12-well plate. After coating, remove excess liquid and wash the inserts
twice with serum-free DMEM or RPMI.
µ
2.
Cell invasion:
Dilute the thawed Matrigel to the desired final concentration using
cooled serum-free medium. Make a 50-
g/mL (II) solution
for 6.5-mm and 12-mm diameter inserts, respectively. Aliquot 100
µ
g/mL (I) and 62.5-
µ
µ
L (I) or
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