APPENDIX I: Media Preparation (M199 and F12K)

1. M199 medium (with and without phenol red - no L-glutamine) and F12K medium (with phenol red - with L- glutamine) were purchased in powdered form from Sigma Co. (Mississauga, ON)

2. The medium were reconstituted in the appropriate volume of water (> 16 Mohms)

3. The reconstituted medium was placed on a hot plate and warmed to 37°C while being constantly stirred

4. Once the medium reached the desired temperature, HEPES was added to make a final concentration of 25 mM (5.95 g/L) and allowed to fully dissolve

5. The pH was then adjusted to 7.2 using 2N NaOH

6. After stabilizing the pH, sodium bicarbonate was added to produce a final concentration of 25 mM (2.0 g/L) and allowed to fully dissolve

7. The medium was then filter sterilized (0.2 µm bottle top filters) into tissue culture media bottles and refrigerated (4°C)

8. When required, medium was removed from the refrigerator and allowed to warm at room temperature

9. M199 was supplemented with L-glutamine (final conc. 200 mM) and gentamycin (final conc. 25 µg/mL) while F12K was supplemented with only gentamycin (final conc. 25 µg/mL)

10.    Fetal bovine was then added to produce the desired final concentration

11.    Media was filtered again (0.2 µm) and warmed to 37°C prior to use