Tissue Culture

NOTE: Due to University Regulations, Tissue Culture and Cell Extraction were observed. When MPP+ was added to the cells, I was not present for safety reasons.

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Materials

• SH-SY5Y neuroblastoma cells
• Minimum essential media
• F12 (Hamilton’s) media
• 10% Fetal bovine serum
• Trypsin-EDTA
• 75 cm2 flask
• Pipette and pipetting apparatus
• 15 ml conical tube
• Centrifuge
• 100 mm cell culturing plates
• Incubator
• Aspirator

Procedure

1. Fresh media for cells was prepared consisting of 1:1 ratio of minimum essential media to F12 (Hamilton’s) media plus 10% Fetal bovine serum.

2. Old media from 75 cm2 flask containing SH-SY5Y cells was pipetted into 15ml conical tube.

3. Two millilitres of trypsin-EDTA was added to the flask.

4. After 2-3 minutes, the cells detached from the bottom of the flask.

5. Three millilitres of fresh media was added to flask to dilute the trypsin-EDTA.

6. Detached cells were pipetted into a 15 ml conical tube which was centrifuged for 5 minutes at 1000 rpm.

7. After centrifuging, remaining old media (supernatant) was aspirated and 10 ml of fresh media was pipetted onto the cell pellet in the 15 ml tube.

8. To break up the cell pellet, the fresh media was pipetted into and out of the tube several times.

9. Hemocytometer count was performed to determine the number of cells in one 15 ml conical tube

10. One million cells were added to each 100 mm plate, each with 10 ml of fresh media.

11. 100 mm dishes were placed in the incubator and left for 4 days.

12. After four days passed, Fetal bovine serum was reduced from 10% to 1% for 24 hours prior to treatments.

13. Treatments were prepared for cells as follows

Guanosine	30 mg of guanosine + 10.5 ml ddH2O + 100µL 1N NaOH = 10 mM stock solution
MPP+	10 mg of + 3.37 ml ddH2O = 10 mM stock solution

14. Old media was aspirated off of cell plates using aspirator. Ten millilitres of fresh media was added to each plate.

15. Guanosine was added for 1 hour prior to exposure with MPP+ for pre-treatment

16. Cells were placed in incubator for 1 hour. After 1 hour had passed, MPP+ was added for induction of apoptosis for 48 hours.

17. For co-treatment, guanosine and MPP+ were added at the same time for 48 hours.

18. For the post-treatment, the guanosine was added after the MPP+.