Proliferation Protocol (Performed twice, once per cell type):

  1. A 1 mL aliquot of cells from a Cryotank was used to inoculate 30 mL of 10% serum DMEM media in a 75mm2 culture flask yielding a cell density of approximately 106 to 2x106 cells.
  1. Thecryo-freezing fluid was replaced with DMEM (10%) and incubated (37° C, 5% CO2) for 2 days until a cell density of approximately 4x106 to 5x106 cells/mL was reached.
  1. The cells were grown up to a density of approximately 1million cells/mL
  1. 0.1 mL of the cell suspension was added to 20 25mm2 tissue culture treated flasks containing 5 mL of DMEM (10%) media, creating a cell density of approximately 105 cells per flask.
  1. The cells were then incubated for the remainder of the day.
  2. The cells were trypsinized and then counted. Nine cell counts were taken per flask, with two flasks per variable group, with 10 flasks per cell type, for a total of 20 flasks.
  1. Both cell types were allowed to re-adhere.
  1. Two flasks from both the 3T3 line and the C2C12 line were exposed to x-rays for .5 seconds at the classical dental x-ray frequency, 90 KVP and 15 ma.
  1. Two flasks from both the 3T3 line and the C2C12 line were then exposed to x-rays for 2 seconds at the classical dental x-ray frequency.
  1. Oxygen was flushed from two flasks from both the 3T3 line and the C2C12 line using N2 tank. Exposure time was 1 second.
  1. Oxygen was again flushed from two other flasks from both the 3T3 line and the C2C12 line using N2 tank, with an exposure time of 10 seconds.
  1. Two flasks of each cell type were left unexposed to serve as respective control groups.
  1. The flasks were then allowed to incubate for 48 hours.
  1. Following incubation, cell counting was repeated as in Step 6.

Differentiation Protocol:

  1. Cells were cultured as in the proliferation assay, and allowed to grow to confluence.
  2. C2C12 cells from the 75mm2 flask were plated onto glass plates with four plates per group, for a total of 10 plates (no ischemic variable group).Cell counts were no taken, but the cells were exposed to oxidative stress as in the proliferation experiment.
  3. Following the application of stress, the 10%DMEM was replaced with 1% DMEM to serum starve the cells and trigger differentiation.
  4. The cells were allowed to differentiate for 3 days before being stained with methanol.
  5. A blocker solution was applied to the glass plates and allowed to bind for 45 minutes.
  6. The blocker was aspirated off and the primary antibody was applied.
  7. The primary antibody was allowed to bind for 45 minutes before being aspirated off and replaced with a secondary antibody stain (myosin).
  8. The myosin stain was allowed to bind for 45 minutes before being aspirated off.
  9. The final antibody, DAPI, was applied and allowed to bind for 3 minutes before being washed off.
  10. Images of each plate were then taken under a fluoroscope.
  11. The percent differentiation was determined using the images and an equation provided by an associate of the Huard Lab, comparing the number of nuclei over all with the number within the myotubes, determined by the glow of the DAPI and myosin, respectively.

References

  1. DiPrimio, Nina, P.h.D. Personal communication. 9 Dec. 2010.
  2. Green, Howard. "The Story of 3T3 Cells." Bio Info. Web. 20 Jan. 2011.
  3. Hill, M. "Cell Biology Lab- Tissue Culture C2C12 Cells." UNSW Cell Biology. Web. 04 Feb. 2011.
  4. Huard, Johnny, P.h.D. Personal communication. 15 Aug. 2010.
  5. Kline, Donald. Personal communication. 5 Jan. 2011.
  6. Krotec, Mark C. Personal communication. Aug.-Feb. 2010.
  7. Staining Protocol. Web. 04 Feb. 2011. <
  8. Zapanta, ConradP.h.D. Personal communication. Nov.-Dec. 2010.