Migration Assays for Oligodendrocyte Precursor Cells - Methods in Molecular Biology: Extracellular Matrix Protocols - page 255

2. m pore polypropylene filter beds—Corning Costar (Cat. No.

155846) ( see Note 2 ).
3. Cells.
4. Growth medium for your cells. For these studies, Sato's modification DMEM
is used.*
5. Bacterial grade plastic Petri dishes (i.e., not tissue culture treated).
6.

Nucleo Pore™ 8- µ

Sterile 10 mL pipets.

7. P1000, P200, P20 variable pipets.

8 Sterile pipet tips.

9. Blunt ended forceps, two pairs.

10. 100% methanol.

11. Eosin, 1% aqueous solution (Merck, Darmstadt, Germany, Cat. No. 35084 4K).

12. Haemotoxylin, Ehrlich's original formulation (Merck Cat. No. 35017 5T).

13. 3 Beakers or staining jars.

14. DePex (Merck Cat. No. 36125 2B).

15. Humidified chamber (e.g., plastic box with wet tissues in the bottom).

16. Phosphate buffered saline (PBS), sterile, tissue culture grade.

17. Protein of interest, e.g., extracellular matrix protein.

18. Chemotactic agent of interest, e.g., growth factor such as platelet-derived growth

factor (PDGF).

19. Poly-D-lysine (PDL) ( Sigma P-7405).

20. 0.5 M acetic acid.

21. Sterile distilled water.

22. Terg-A-Zyme (Alconox, Inc., New York) or other nonaggressive enzyme cleaner.

Not Decon.

2.2. Agarose Drop Migration Assay
1.

Low melting point agarose (Sigma A-9045).

2. Phosphate buffered saline (PBS) sterile, tissue culture grade.

3. Microwave oven.

4. Water bath set at 37 ° C.

5. Cells.

*Sato's modification of DMEM for oligodendrocyte precursor migration assay. There are
several versions of Sato's modification of DMEM. The following recipe has been used in this lab
for 5 yr without problems:
Dulbecco's Modified Eagle's Medium, high glucose with pyruvate (DMEM) (Sigma D-6546);
Penicillin/Streptomycin solution f.c. 100 U/mL (Life Technologies - Cat. No. 15140 114);
L -Glutamine (Sigma G-6392) f.c. 4 m M ; Putrescine (Sigma P-5780) f.c. 16 g/mL; Thyroxine

(Sigma (T4) T-1775) f.c. 400 µg/mL; Tri-Iodothyroxine (Sigma (T3) T-6397) f.c. 400 µg/mL;
Progesterone (Sigma P-8783) f.c. 66 ng/mL; Sodium Selenite (Sigma S-5261) f.c. 5 ng/mL;
Bovine Serum Albumin (BSA) Fraction V (Sigma A-4919) f.c. 100 µg/mL; Insulin (Sigma I-1882)
f.c. 5 µg/mL; holo-Transferrin (human) (Sigma T-0665) f.c. 50 µg/mL. T3 and T4 do not dissolve
in water/DMEM, they need to be solubilized in 0.1 M NaOH, and 0.2 M NaOH, or 4.0 M NH 4 OH
in 75% MetOH, respectively.

µ