- Abstract
- Introduction
- Materials
- Methods
- Detailed Methods
- Results
- Calculations
- Discussion and Relevance
- Conclusion
- References
- Acknowledgements
- Project Info
A) Cell culture
On day 1, ShyY cells for the different assays were plated from stock cells grown in flasks utilizing differing growth media (MEM, HS, FBS, P/S). Plated cells at 10^5 (ten to the five) cells/ml in wells were coated with collagen substrate were then incubated with fresh growth media containing 100 ng/ml of 7S NGF for a further 6 (six) days. Cells were then fed fresh NGF-containing growth medium every 2 (two) days.
B) PC12 Cell Survival Assay using Zapoglobin Cell Lysis
Washed ShyC cells with fresh medium, and incubated the cells in growth medium plus NGF/500uM Hydrogen peroxide with 0.1-10ml blueberry extract. The cells were then harvested from the wells 24 hours after they had been growing under above conditions, while HBSS was added, and gentle pipetting was used to dislodge the cells. Cells from wells were then spun down tubes in bench microcentrifuge and suernatant was discarded. Cells were lyced with 200ul zapoglobin cell lysis solution in HBSS. Viable cells were counted.
C) Viability Staining with Ethidium Bromide and Acridine Oraage (Hoechst stain)
Cells were washed with fresh medium, and incubated as above. Next, they were again harvested (with HBSS harvested and gentil pipeetting to dislodge cells) 24 hours after they had been growing. Cells from wells were then spun down eppendorf tubes in bench microcentrifuge, and supernatant was discard. Cells were resuspended in 1 ml HBSS with an estimated 1-5x10^5 cells/ml. 25Ul volumes of cell suspension and ethidium bromide orange (Hoechst stain) to an ependorf tube, and it was gently mixed. An aliquot (25ul) was placed underneath coverslip on hemocytometer slide. cells were observed under 100x and 400x. Cells were counted after switching to fluorescent lighting..
D) Cell Proliferation Kit Procedure (MTT ASSAY)
Cells were gown in 96 well, flat bottomed plates each having a final volume of 100 microlitres of culture. The cells were grown in the humidified environment of a incubator set at 35 degrees Celsius and 6.5% carbon dioxide for 24 to 96 hours. When the incubation period expired 10 microlitres of the MTT labeling reagent was added to each well. The wells were incubated for 4 hours at 35 degrees Celsius with a 6.5% carbon dioxide atmosphere. One hundred microlitres of stabilization solution was added to each well and the plate was incubated overnight at 35 degrees Celsius with a 6.5% carbon dioxide atmosphere. Complete solubilization of the formazan crystals was ensured and the spectrophotometrical absorbance of the samples was checked using a microtitercal (ELISA) reader.
Copyright 2004 Gabriel Rigg, Michael
Armson, David Dickinson.
All Rights reserved