Biology Reference
In-Depth Information
3.
Obtain the cells for investigation, by overnight mechanical shaking of mixed glial
cultures for oligodendrocyte precursors, centrifuge to a pellet, at 240
g
for 5 min.
4.
Remove the supernatant and resuspend the pellet in 40
L of Sato's medium
containing FCS (or HS), triturate up and down vigorously several times before
transferring exactly 40
µ
L of the cell suspension to a clean microfuge tube
(
see
Note 26
). The cell density used varied between 20 and 100
µ
×
10
6
cells/mL
(
see
Note 27
).
5.
Transfer the tube with the cell suspension in to the 37
C water bath to equilibrate
the temperature. After 2 min or so, transfer the tube to a plastic beaker containing
37
°
C water from the bath, to maintain the temperature during subsequent
handling in the TC hood. Add 20
°
µ
L of the 1% agarose to the cells and mix
thoroughly.
6.
Immediately, plate 1.5
L drops of the cell suspension at the centre of wells in a
24-well tissue culture plate. Place the plate in a refrigerator at 4
µ
°
C for 15 min to
allow the agarose drop to set (
see
Notes 28
and
29
).
7.
As the drops are setting, prepare the solutions to be added around the drop. In the
case of migration on PDL, the solution to be added would be Sato's medium
alone. However, to examine cell migration on different ECM molecules, stock
solutions of ECM should be diluted into Sato's medium to give a final concentra-
tion of 10
µ
g/mL.
8.
After the drop has been at 4
L Sato's medium
media (+/- ECM) around the drop (
see
Note 30
). When ECM solution is being
used, the dish should then be placed into the incubator at 37
°
C for 15 min, carefully add 50
µ
°
C for 2 h in order for
the ECM solution to coat the plastic surface.
9.
After 2 h, a further 450
L of Sato's medium can be carefully added (down the
side of the well) before returning the plates to the incubator (
see
Note 31
).
µ
10.
The extent of cell migration can then be measured at daily intervals for 1-5 d
using a phase contrast microscope with a calibrated graticule in the eyepiece, in
which the width of one grid square represents 100
µ
m actual distance at a magni-
fication of 10
. Cells migrate out to form a uniform corona around the drop (
see
Fig. 1
). At any one time point, the distance between the edge of the drop and the
leading edge of migrating cells within the corona can be recorded on four sides of
the drop. We routinely count at 0, 90, 180, and 270
×
angles around the drop (
see
Note 32
). A distribution curve can be established by measuring the number of
cells at various distances from the edge of the agarose drop.
°
11.
The cells should be allowed to migrate out of the drop for 24 h before any func-
tion blocking reagents are introduced. This allows direct analysis of the inhibi-
tion of cell migration per se, as opposed to the reagent interfering with the ability
of the cells to exit from the agarose drop. The inhibitors are added carefully,
down the side of the wells, in volumes of 5-20
L.
12. In order to discount cell proliferation as a factor in this assay, aphidicolin at
20
µ
L/mL can be included in the surrounding media. Aphidicolin inhibits DNA
replication, and thus the contribution of cell proliferation to the expanding corona
of cells around the drop can be discounted
(11)
.
µ
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