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Cell Culture Preparation
Preparation of Meloxicam Solution
Addition of Meloxicam to Cell Lines
MTT Assay
Apoptosis Assay
Western Blot
 
 
Cell Culture Preparation
          The 184-A1, HTB-129 and HTB-132 cells were incubated at 37 C under 5% CO₂. When each cell line was almost 80% confluent, the cells were split into 24 well plates, 24 hours prior to the addition of the meloxicam. 4.00 mL DMEM solution were added to each well to allow the cells to equilibrate to the new conditions. By splitting the cells into 24 well plates, each concentration could be done in triplicate, so that there was a sufficiently large sample size, which would give more reliable results where percentage error could be calculated.  
 
 

Cells were incubated in this incubator at 37°C under 5% CO2.

The following layout was used when placing cells into 24 well plates.

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Preparation of Meloxicam Solution
          1.50 mg of meloxicam was dissolved into 7.40 mL DMEM solution to obtain a 0.54 µmol meloxicam solution. 5.00mL of the 0.54 µmol meloxicam solution was mixed with 1.00 mL DMEM solution to obtain a 0.36 µmol solution. 2.50 mL of 0.36 µmol meloxicam solution was mixed with 2.50 mL DMEM solution to obtain a 0.18 µmol solution.
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Addition of Meloxicam to Cell Lines
          To the first column of cells in each plate, 4.0 µmol of DMEM solution was added. These were the negative control group. To the second column of cells in each plate, 4.00 µL 0.18 µmol meloxicam solution was added. To the third column of cells in each plate, 4.00 µL 0.36 µmol meloxicam solution was added. To the fourth column of cells in each plate, 4.00 µL of 0.54 µmol meloxicam solution was added. After 24 hours, the medium from each well plate was removed using a vacuum.
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MTT Assay
          In order to see how much cell death/growth had taken place for each cell line under each concentration of meloxicam, an MTT assay was performed. 3.0 g of the MTT dye, thiozolyl blue (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) was dissolved in 3.60 mL PBS solution and 0.40 mL DMEM. The DMEM would provide nutrients for any living cells so that they would not die. The PBS solution provided an isotonic environment that the cells can survive in.
          2.0mL of the PBS with dissolved thiozolyl blue and DMEM was added to each well plate. The cells were then allowed to sit in the incubator for 1 hour. Any living cells would take in the MTT dye and reduce it to a blue-black formazan crystal in their mitochondria. Dead cells would not do this. Thus, the more living cells in each well, the more formazan crystal would be produced, giving the well a more intense purple colour.
          100 µL of 50% dimethylformamide (DMF) was added to each well and the well plate was allowed to sit for 5 hours. DMF lysed the cells and dissolved the formazan crystals, giving the wells a blue/purple colour. 1.00mL of the well's contents was transferred into a 96 well plate. The 96 well plate was placed in an ELISA plate reader and the dye's optical density was read at 590 nm, producing a comparable quantitative value for each well. The percentage viability of the cell culture in each well could now be calculated using the formula: Viability of well = 100% × (O.D. of well/ O.D.of control well).
 
 

A picture of me transferring aliquot samples for the MTT analysis.

Well plates with the purple MTT dye in them.

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Apoptosis Assay
          The apoptosis assay showed whether apoptosis was taking place in the cell lines.
          First, contents of each well were gently vacuumed out. 3 mL of 10% PBS solution with 5% APOPercentage assay dye was added to each well. The cells were allowed to incubate for 20 minutes. Pictures of the wells were taken at this point. The contents of the wells were then gently vacuumed out. The cells were then lysed using DMF. The redder a well was, the more cells had committed apoptosis in the well. Aliquot samples of each well in the 24 well plate were transferred to a 96 well plate. The optical density of each well was then read using an ELISA plate reader.
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Western Blot
          The western blot analysis would show how much bcl-2 was present in each cell line after exposure to different concentrations of the COX-2 inhibitor, meloxicam.
          The proteins of each cell culture was separated using SDS-polyacrylamide gel electrophoresis. Electrophoresis separated the proteins by size. Larger proteins remained closer to the wells than proteins of smaller size. Bcl-2, with a size of 25 kDa. A nitrocellulose membrane was placed on the gel and once again, using electrophoresis the polypeptide bands on the gel were driven onto the extremely adhesive nitrocellulose membrane.
          The nitrocellulose membrane was then incubated for 24 hours with a primary antibody that was specific for the bcl-2 protein. The primary antibody, attached to the bcl-2 protein and formed an antibody-protein complex.
          Then, the nitrocellulose membrane was incubated with a secondary antibody. This antibody was an antibody-enzyme conjugate. It was specific for the anti-bcl-2 antibody. A detection solution consisting of horseradish peroxidase was added onto the nitrocellulose membrane. The membrane was then placed in a protective transparent envelope and allowed to expose an X-ray film for approximately 5 minutes, producing exposed bands on the film wherever high levels of bcl-2 was present.
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