Michael, Entry #2, Moving Along

I have come to realize that 8 weeks in lab really isn't much time. I am currently at the start of my fifth week, and due to the necessary prep work and mastery of techniques needed for my experiments to succeed, I am just now on the cusp of running my first trials. In addition to maintaining plates of HepG2 cells, I, along with the two other high school students (Jalen and Sidney) have transfected these cells. After doing two practice transfections, we realized we would have to do another before beginning our research because the bottle of the "Extreme Gene" that we used was almost empty and we would have to open a new one. Since the optimal ratio of "Extreme Gene" to GFP (fluorescent plasmid) varies from bottle to bottle, another transfection was necessary to determine this ratio.


When we came in to check on the transfection the day after, Jalen, Sidney, and I realized that all of our cells were contaminated (the transfection plates and stock plates). It was the first time this happened, and it will hopefully be the last. We all had to obtain new HepG2 cells to maintain our stock, but this was just a matter of borrowing cells from Sumedha, who had extra plates to give us.


The worst part about the contamination was that it could cause our transfection to fail, meaning we would have to do yet another, which would set us back almost a week (due to the process of growing the cells, splitting them, transfecting, and determining the efficiency). However, when we looked at the transfection under the GFP-sensitive microscope, we saw that the plasmid was inserted into some cells, and that the 3:1 ratio worked better than the 2:1 ratio. This was the main question we needed to answer; therefore, the transfection was a success and we could move on to the next step of our respective projects.


My next step was to learn how to measure glucose uptake in HepG2 cells. I used a six-well plate of cells in glucose-free media. I added nothing to two of the wells (C), added fluorescent glucose to two (G), and added glucose and insulin to the last pair (GI). After two hours, I collected the cells, spun them down in the centrifuge, and transferred them to special tubes to be used in the Guava machine, which determined how much glucose the cells took up by measuring fluorescence.

The readings from the Guava machine, which show how the different wells differ.

The Guava machine showed that the G and GI wells took up significantly more glucose than the C wells. However, we expected the GI wells to take up more glucose than the G wells (because insulin facilitates glucose uptake), but this did not occur. My PI suggested that, since HepG2 cells are a cancer cell line, and cancer cell lines take up much more glucose than normal cells, the insulin might not have had an effect on these cells. The plan is to perform the same experiment in HepG2 cells and primary rat hepatocytes (which aren't a cancer cell line) and to determine which cells are better suited for my project. After we determine this, I will experiment with HBV-infected cells, to see if and how the virus regulates glucose uptake.

The Guava machine itself. The silver box is where the tubes are inserted, and the data is projected on the laptop.