Biology Reference
In-Depth Information
250
L (II) of solution into the upper surface of the membrane of the inserts.
Make sure to keep all undiluted solutions on ice to avoid undesired gelification.
Leave the plates in a cell-culture hood to allow the Matrigel solution to air-dry. It
takes about 5 h to dehydrate the indicated amount of Matrigel under a continuous
laminar flow. For convenience, it is possible to leave the inserts to air-dry over-
night in the absence of laminar flow. Reconstitute Matrigel with 100-200
µ
L of
serum-free DMEM or RPMI at room temperature for 90 min under constant rota-
tion. Remove the excess medium from the membranes before adding the cells.
µ
3.2.2. Cell Labeling
1.
When assaying anchorage-dependent cells, rinse the adherent cells with PBS, add
5 m
M
EDTA in PBS and incubate at 37
°
C in 5% CO
2
for 2-5 min (
see
Note 18
).
2.
Collect the detached cells and wash them twice in serum-free DMEM. When
assaying suspension-growing cells, collect the cells directly from the flask and
wash them twice with serum-free RPMI.
3.
Prepare the working dye solution by diluting the concentrated stock solution in
0.25
M
sucrose to reach the final concentration of 1-10
µ
M
(
see
Note 18
).
10
6
cells) in 200
4.
Resuspend the cell pellet (
≤
5
×
µ
L of dye solution.
5.
Incubate the cell suspension at 37
°
C in 5 % CO
2
for 30-45 min (
see
Note 19
).
6.
Wash the cells twice with the working assay solution.
3.2.3. Procedure for FATIMA Using Unicell 24 Plates (
Fig. 3
)
1.
10
6
cells/mL (leukocytes), or at
Resuspend the cells in working solution at 2
×
10
6
cells/mL (anchorage-dependent) and fill the upper portion of the mem-
brane with 100
1
×
µ
L (for 6.5-mm diameter inserts) or 500
µ
L (for 12-mm diameter
inserts) of the cell suspension.
2.
Fill the lower part of the unit, i.e., the 24-well plate using as a tray for the
Unicell 24 plate through the openings in the insert wall, with 600
L (6.5 mm) or
1.5 mL (12 mm) of control medium (working assay solution); conditioned
medium (as a positive control); and/or other potential stimulating agents diluted
in working assay solution.
µ
3.
Incubate the plates at 37
°
C in 5% CO
2
for the desired time (
see
Note 20
).
4.
At the end of the assay, or at the desired time-intervals, and without manipulating
the inserts, determine the fluorescence levels using the microplate reader. The
detected fluorescence value corresponds to the total number of cells (
Fig. 3
).
5.
Remove the nonmigratory/noninvading cells from the upper surface of the mem-
brane with a cotton swab (
Fig. 3
). Perform this operation for anchorage-depen-
dent cells assuring to dislodge all cells residing near the border of the insert,
without removing the inserts from their original well. For suspension-growing
cells, removal of nonmigratory cells from the upper side of the membrane is not
necessary since the 24-insert Unicell 24 unit, can temporary be transferred to an
adjacent empty well or to another plate (
Fig. 3
).
6.
Measure the fluorescence levels (in the microplate reader), which now corre-
sponds to the migrated cells (
Fig. 3
).
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