- 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
1.Washed cells with fresh Medium
2.Incubated cells in growth medium plus NGF/*dose H2O2 together with 0.1-10 mg/ml blueberry extract.
3.Harvested cells from wells (added HBSS to well and used gentle pipetting to dislodge cells) 24 hrs after they had been growing under the above conditions.
4.Spun cells from wells down in tubes in bench microcentrifuge and discarded supernatant.
5.Resuspended cells with 200 microliter zapoglobin cell lysis solution in HBSS.
6.Counted viable nuclei using hemocytometer and microscope (intact nuclei from viable cells are phase-dense) to determine the number of viable cells/ml and the total number cells that had survived the assay conditions.
* Dose of H2O2 reducing cell viability by 50% was determined.
C. Viability Staining with Ethidium Bromide and Acridine Orange
1.Washed cells with fresh medium
2.Incubateed cells in growth medium plus NGF/*dose H2O2 together with 0.1-10 mg/ml blueberry extract.
3.Harvested cells from wells (added HBSS to well and used gentle pipetting to dislodge cells) 24 hrs after they had been growing under the above conditions.
4.Spun cells from wells down in eppendorf tubes in bench microcentrifuge and discarded supernatant, resuspended cell in 1 ml HBSS with an estimated 1-5 x 105 cells/ml
5.Added equal 25 microliter volumes of cell suspension and ethidium bromide/acridine orange to an ependorf tube. Mixed gently
6.Placed an aliquot (aprox. 25 microliter) underneath the coverslip on the hemocytometer slide.
7.Observed cells initially under the microscope using visible light, 100-400X. Adjusted diaphram to reduce light and keep hemocytometer grid visible.
8.Keeping visible light on switch to fluorescence and observe cells. Live cells fluoresced green (with acridine orange) and dead cell fluoresced orange (with ethidium bromide).
9.Counted cells and calculated viability and number of apoptotic versus necrotic cells.
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