Isolating bone marrow from mice, an experiment that requires the teamwork of the entire lab.

Author: Danica McGrevey Major: Biology

I conduct immunology research under Dr. Jeannine Durdik in the biological sciences department in the Fulbright College of Arts and Sciences. Dr. Durdik’s lab is interested in the effect of fever on the immune response. Fever stimulates both innate and adaptive immune responses and is therefore vital in fighting disease. My project specifically uses rainbow trout and the RTS11 cell line derived from the spleen of rainbow trout as a model organism to analyze the effect of fever temperature on immune response. This semester I made great strides in my research, completing one method of experimentation and training for a new method to begin at the start of 2021. This project has given me a great introduction to the research world and has given me an abundance of knowledge about the immune system, which I will absolutely use in my future career as I begin medical school in July 2021.

This semester I worked with the RTS11 cell line, as well as assisted the lab in additional projects involving fever temperatures in order to both learn research techniques and to be a helpful hand to those who have helped me so greatly in my research endeavors. My project specifically uses macrophages, immune system cells that are important for initial immune response, from rainbow trout fish head kidney and the RTS11 cell line.

This year I conducted experiments in which I measured the nitric oxide production of macrophages.Macrophages produce nitric oxide (NO) as a highly reactive molecule with the potential to destroy pathogens and to signal to other cells that defenses are needed. I incubated stimulated macrophages at a variety of temperatures with the goal of finding the temperature at which the macrophages responded best. I analyzed the results by using a Griess Reagent to convert NO to nitrites, which I then measured in order to quantify and compare the activity of macrophages at different temperatures.

When I began this research in Spring 2020, I stimulated macrophage activity with lipopolysaccharide (LPS), a component of the cell walls of gram-negative bacteria, inducing an immune response. However, even with multiple trials of different concentrations of LPS, I did not have successful stimulation. This semester I began to work with additional stimulants: poly I:C, an analog of double stranded RNA that serves as an immunostimulant, and peptidoglycan, a component of the cell walls of bacteria. These two stimulants proved better able to stimulate RTS11 than the initial stimulant selection, LPS.

Another barrier to successful stimulation involved the media in which the RTS11 cell line could be successfully grown. With the help of Dr. Durdik and Intisar Hassan, a PhD student in the lab who has helped me greatly, we discovered that a component of the media needed for growth of RTS11, fetal bovine serum (FBS), could be conflicting with stimulation. We decided to try media with varying concentrations of FBS to see if I could obtain better results. We found that using 0.1% FBS allows for successful stimulation, whereas 10% FBS is necessary for growth and routine culture of the RTS11 cell line.

I initially incubated the macrophages for 24 hours; however, I began to increase this time when I did not obtain strong results. This semester I found that macrophages respond best at 48 hours. Additionally, I found a result which I was not initially searching for, but that I have unintentionally supported with many trials of my experiments. At higher temperatures, macrophages produce high levels of nitric oxide over time, even without stimulation. This finding emphasizes the importance of fever alone in the immune response.

In Spring 2021, I will continue my project with a second method of experimentation: protein synthesis. My lab is also working to obtain rainbow trout so that I may obtain more data with the rainbow trout head kidney macrophages. As I prepare to complete my project and defend my resulting thesis in the midst of a pandemic, I am acutely aware of the importance of better understanding the role of fever in fighting disease.