By Cole Hughes, Fabian Torres, and Dustin Bullock
Brescia University
717 Frederica St, 42301
Molecular Cell Biology Lab
OUR PROPOSITION
Methyl Alpha-D-Glucose will target the energy metabolism of the cancer cell. Autophagy is the degradation of cytoplasmic components within lysosomes. In the experiment we will be adding methyl alpha-D-Glucose to the HeLa cells. We hypothesize this will inhibit the growth by targeting the metabolic processes of the cells.
METHODS
3/19/18 Day 1:
1430: Start of experiment the HeLa cells were stored at 37 degrees Celsius with 5 percent Co2. We used 2 wells the top was the control and the bottom was drug. Â The HeLa cells were pipetted into all 4 cells.We used Methyl Alpha-D-Glucose as our drug. we used a concentration of 100 micro molar. We pipetted 30 micro-liters of methyl into the bottom 2 wells. The plate with the HeLa cells was set to incubate for 24 hours at 37 degrees with 5 percent Co2.Â
3/20/18 Day2:Â
LC3/Methanol fixation
1100: Remove plate from incubator. We removed 300ml of the media and added 400ul of methanol to the HeLa cells. The plate was placed in a freezer at -20 degrees Celsius. it will cool for 10 minutes. After the 10 minutes of cooling the 400ul of methanol was removed. The HeLa cells will sit at room temp of 74 degrees Fahrenheit for 3 minutes. 400ul of a permeabilization buffer Tween 20 was added to the HeLa cells. The HeLa cells will sit at room temperature for 10 minutes. Now add a few drops of enhancer goat serum to the HeLa cells and 400ul of a blocking buffer was added. Incubate for 24 hours
3/27/18 Day 3:
1206: 3 percent goat serum 2ul of LC3. 200ul of this solution was added to the control and to the drug cell. we let it sit at room temperature for 2 hours. Â Â Â
1400:Â We added 200ul of the goat serum to the HeLa cells. The HeLa cells were placed in a dark room for 30 minutes. 200ul of DAPI stain was added into the cells. The HeLa cells were incubated for 8 minutes at room temperature in a dark room.
1509: The wells were removed and the slide was cleaned in pure water. After it dried, 1 drop of Prolong Gold Mounting Medium was added to each well.
1512: The slide was placed in a dark room to sit over night.
FIGURES
First Image- Hela Cells Drug Group. Visualized with a 40X Objective. LC3 Antibody (Red) Stained with DAPI (Blue) Second Image- HeLa Cells Controlled Group. Visualized with a 40X objective. LC3 Antibody (red) stained with DAPI (Blue)
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RESULTS
Our results were inconclusive due to the limited visibility of our cells when we evaluated our cells under a microscope. It was evident that that there were cells present, but not enough cells were presented with the DAPI stain making it much harder to obtain quality images for both the the controlled well and the drug induced well. There was still some areas that did properly obtain the DAPI stain which is why were were able to obtain the images that we did, but there should have been a more abundance of staining all thorough out the well plates. The possible flaws could reside in our staining protocol. In the dilution process of our DAPI stain involved the incubation period of 8 minutes after applying our stain, if we incubated the stain for less than 8 minutes, that could have influenced how visible our cells were. Another issue could have been the actual addition of our DAPI stain, because there was a possibility of flooding our cells if were weren't gentle when micropipetting our DAPI stain onto our wells.
CONCLUSION
We chose Methyl alpha d glucose because it was what we thought would be the best possible drug to use to make the cells go through autophagy. It makes for an interesting topic because we gain knowledge on how caner cells can react to different conditions. We believe that methyl alpha d glucose will inhibit the growth of cancer and kill of the cancer cells thus making a more affective cancer treatment, this adds to the greater scheme of life in the aspect that new ideas can aid in the process of understanding a complex process like cancer. we have came across several problems while doing our experiment. These problems are, inconsistency's with the pipetting, not every thing could have been removed from the HeLa Cells that should have. Contamination, and some of the steps were rushed due to time complication. These problems could be solved by having only one person pipet, and don't rush some of the steps.
ACKNOWLEDGEMENTS
We would like to thank Dr. Jacob Adler and Brescia University for providing us with the facility and equipment to conduct our experiment. We would also like to thank Title III Strengthening Institutions Grant for covering expences.
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