Biology Reference
In-Depth Information
14.
Place the chamber in a humidified chamber at 37
°
C until you are ready to put the
cells in (
see
Note 14
).
15.
Remove the cell suspension from the Petri dishes and centrifuge at 240
g
for 5 min
(
see
also
Note 7
).
16.
Remove supernatant and resuspend the cells in growth medium.
17.
Triturate the cells for 30 s, rest for 30 s, and retriturate, repeating this three times
(
see
Note 15
).
18.
Take a small aliquot and count the cells.
19.
Dilute the cells to achieve 35-40,000 cells per 50
µ
L, and add any test solution to
the cells.
20.
Pipet 50
L cells into each of the wells in the upper plate, taking care to avoid any
bubbles forming on the top of the filter (
see
Note 16
).
µ
21.
Replace in the humidified chamber, at 37
°
C, for the requisite period of time, 8 or
16 h (
see
Note 17
).
22.
At the completion of the experiment, carefully remove the thumb nuts, lift the
top plate off the chamber, and using forceps place the filter in 100% methanol for
2 min to fix the cells (
see
Notes 18-20
).
23.
Stain with eosin for 2 min.
24.
Stain with haemotoxylin for 1 min.
25.
Rinse in distilled water, then differentiate the stain in tap water for 10-20 s, then
rinse in distilled water.
26.
Place the filter shiny side down on a clean microscope slide taking care to avoid
the filter folding during handling (
see
Note 21
).
27.
Wipe the unmigrated cells off the top of the filter using a wet cotton bud (
see
Note 22
).
28.
Dry the filter using a dry cotton bud or a tissue.
29.
Mount on DePex.
30.
Count the cells using a light microscope (
see
Notes 23
and
24
).
3.2. Agarose Drop Migration Assay
For the purposes of this description the cell type in use is the oligodendro-
cyte precursor cell, for which the growth medium would be Sato's modifica-
tion of DMEM
(7)
.
1.
Make a solution of 1% agarose, for example, 50 mg of agarose in 5 mL of PBS,
and dissolve in a microwave oven. When dissolving agarose, you need to micro-
wave it for a few short periods of time, making sure that you do not overheat the
mixture, and mix between each period of heating. Once the agarose is fully dis-
solved, the tube can be placed in a 37
°
C water bath to prevent premature gelling
of the agarose (
see
Note 25
).
2.
Make up the working solutions for resuspending the cells, e.g., Sato's medium
plus 10% FBS, for rat oligodendrocyte precursor cells or horse serum (HS), for
mouse oligodendrocyte precursor cells, and warm to 37
°
C in the water bath prior
to the experiment.
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