Migration Assays for Oligodendrocyte Precursor Cells - Methods in Molecular Biology: Extracellular Matrix Protocols - page 256

6. Growth medium for your cells. ( See Subheading 2.1.4. )

7. Variable pipets, P20, P100, and P1000.

8. Sterile pipet tips.

9. 24 Multiwell tissue culture treated plates (e.g., Nunc, Wiesbaden-biebrich, Ger-

many, Cat. No. 143982).

10. Fetal bovine serum (FBS) for rat derived oligodendrocytes, or Horse Serum (HS)

for mouse derived cells.

11. Sterile microfuge tubes (1.5 mL volume).

12. Plastic beaker.

13. Protein of interest, e.g., extracellular matrix protein or growth factor.

3. Methods
3.1. The Chemotaxis Chamber
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. Soak the filter in 0.5 M acetic acid for 40 min at 60 ° C, or overnight at room

temperature

2. Carefully, wash the filter twice in sterile distilled water ( see Notes 2 and 3 ).

3. Precoat with 5 µ g/mL PDL for a minimum of 1 h ( see Notes 4 ).

4. Wash the filter carefully, twice, in sterile distilled water.

5. Dry the filter ( see Note 5 ).

6. If coating the filter with ECM protein, place shiny side down on a drop of ECM

protein in PBS, and on a clean microscope slide in a humidified chamber, at
37 C; for a minimum of 1 h, preferably overnight. Carefully, rinse the filter in

PBS before placing onto chamber ( see Note 6 ).

°

7. Subtract microglia from shaken cells, by differential adhesion to nontissue cul-

ture treated plastic, for 20-25 min ( see Note 7 ).

8. During which time, prepare the solutions for the chamber, e.g., PDGF dose

response curve ± other factors.

9. Pipet-test solutions into the bottom plate of the chemotaxis chamber ( see Notes 8

and 9 ).

10. Snip a corner off the filter to allow for orientation after staining. Once the filter is

stained, it is virtually impossible to reorientate it correctly without snipping off a
corner. Orientation of the filter is required to not only identify which side to wipe
the cells off, but also to identify which wells are which ( see Note 10 ).

11. Carefully place the filter shiny side down on the bottom plate making sure to not

move the filter once it is placed, otherwise, you might cross-contaminate the test
solutions ( see Note 11 ).

12. Carefully place the silicon gasket on top of the filter ( see Note 12 ).

13. Place the top plate on the chamber, press down with one hand, and maintain even

pressure to avoid bubbles that can form in the lower plate. Then, screw down the
thumb nuts, making sure that all are as finger tight as possible ( see Note 13 ).