Procedure:

         Experimentation on the HepG2 cell line was completed in a McGill University laboratory, in the Division of Cardiovascular Research.
         Experimenation on the MCF-7 cell line was completed in a McGill University laboratory, in the Division of Basic Cancer Research & Oncology.
 
         In order to test my hypotheses, I needed to expose my cultured HepG2 (human liver cancer cell line) and MCF-7 (human breast cancer cell line) cells to different doses of CLAs.
Step #:
Description
   
1 Preparation of conjugated linoleic acid and oleate:
         28.05 mg of CLA was dissolved in ethanol and then evaporated under Nitrogen gas. Next, CLA was dissolved in hot water and then added to a phosphate buffered saline (PBS) solution, containing 2.5 mM bovine serum albumin (BSA), to form a 10 mM stock solution of CLA. Oleate, a common fatty acid, was used as a negative control and a similar procedure was followed to make a 10 mM stock solution of Oleate. Aliquots of this stock solution were stored under nitrogen at -70 degrees until needed.
   
2 Dilution of sample substances:
         Test medium containing the appropriate fatty acid concentrations (CLA and oleate are a fatty acids) were made as follows. For the 50 µM CLA, 100 µl of the stock solution was added to 19.9 ml of serum-free minimum essential medium. Similarly, the 100 µM CLA and Oleate were made by adding 19.8 ml of medium to 200 µl of the stock solution. Next, the 200 µM CLA was made by mixing 400 µl of the stock solution with 19.6 ml of the medium.
   
3 Preparation of cell cultures:
         HepG2 cultures were set up as follows. Forty thousand cells per well were placed in twenty-four well plates containing Minimal Essential Medium with 10% Fetal Calf Serum. After 24 hours, the medium was replaced with serum-free medium for 24 hours.
   
4 Application of test substances to cell cultures:
         The old medium was removed and medium with the different concentrations of CLA, Oleate or serum-free medium alone were added to the cells. All samples were done in hexplicate (sixes). Because there were two separate time points (24 and 48 hours), two groups of samples were set up. Each time point required one 24 well plate. Thus a total of 48 samples, including controls, was tested. The first plate will be examined after 24 hours, and the second plate after 48 hours.
   
5 Incubation of cell cultures:
         Cell cultures were incubated at 37 degrees celcius under 5% CO2 for 24 and 48 hours respectively.
   
   
6 - A Performing cell viability assay - transferring samples:
         Cell viability of all samples was examined using a modified MTT (3-(4,5-dimethylthiaxol-2-yl)-2,5-diphenyltetrazolium bromide) assay and a CytoTox 96 Cytotoxicity assay. 100µl of each sample was pipetted into a 96 well plate.
   
6 - B Performing cell viability assay - adding the reagent:
         Next, 20µl of the manufacturer's reagent was added to each well.
   
6 - C Performing cell viability assay - waiting for color change:
        Approximately 45 minutes of waiting time was required before a significant color change occured, which indicates that the assay is working.
   
6 - D Performing cell viability assay - measuring absorbency:
         Immidiately, the absorbency (optical density) of each sample was read using a spectrophotometer. The percentage cell death (cytotoxicity) was calculated using the formula: 100 - ((Average optical density of sample / Average optical density of negative controls) x 100). This formula makes the 0% point the negative controls.