Rodney Till and Dr. Brian Poole, Department of Microbiology and Molecular Biology

My project this year was a great learning experience and helped me to better understand the complexity of lab work, as well as, the tools used in troubleshooting problems; including collaboration with lab researchers and professors. I learned about flow cytometry and how to analyze migration data. I also learned how to do transfections with HEK cells and how to separate B cells from a fresh sample of blood. I was thankful that I could spend time doing lab work because it’s the best teaching environment I know of in academia. I came across genuine problems and studied topics with the intent of using my knowledge to inform my research and legitimize my experiments.

I would recommend the experience to others as I was happy about the funding that I received and that it allowed me to purchase plasmids and chemokine necessary for the migration study. It’s really not possible to do research of any value without funding and I’m grateful for those would contribute to the ORCA grants. The mentorship programs here at BYU are unique in that there are so many opportunities and that the professors that I’ve met have largely a good attitude toward undergraduates who take research seriously and who are willing to work hard and learn from their mistakes.

During the course of this year this project has been met with many setbacks. For example I began with the hope that the B-cell migration experiment controls would be very easy to establish, but was perplexed with the setup of the transwell plates when the Ramos cells chosen did not migrate toward the chemokine gradient. In response we ran a migration with Ramos, LCL, and Primary B-cells (from extracted blood) only to find that the Ramos cells didn’t migrate at all. We had previously checked the pore sizes of the plates and the chemokine concentrations collaboration with Dr. Wilson who does a lot of migration studies often with
mouse cells, but it turns out that the Ramos cells were of the wrong type.

After determining that the Primary B-cells would migrate we checked to see that we had IRB approval and then we began drawing blood from volunteers and extracting their Primary B-cells in order to use in the migration experiments. Thankfully I’m a certified Phlebotomist and this was an easy process and each volunteer (largely from our lab) signed wavers before the blood draws. After a few weeks of study though, we found out that the Primary cells don’t stay viable for more than a few days which was likely the reason these new cells didn’t migrate after a few initial migration experiments as they were performed after the initial two days following extraction.

In order to coordinate all of the steps involved in the project we set up a schedule so that every part of the experiment from drawing blood and infecting the cells, to running the migration and analyzing the data could be done in the correct order. This time table was limited by weekends and the scheduling of the RIC facility; which is where the equipment for cell counting and migration analysis is located. The largest limiting factor though was experimental error which could derail the process at any point forcing us to begin again on the next appropriate start date. This really slowed down the process. Protocols were updated and created and Dr. Poole was consulted about whether our ideas related to troubleshooting were likely based on correct assumptions, and how we might avoid wasting time looking in the wrong places for error.

Some of the challenges and time-consuming aspects related to this project were: our viral titers were too low and we currently still need to use the viral centrifuge in Dr. McCleary’s lab in order to get a higher titer, refining the protocol for our transfections which had dramatic results, the choice to change plasmids from P-Ultra to a P-Ultra “hot” (which fluoresces red) and the choice to use a cell line that when infected with EBV turns green, in addition the blunt end ligation related to the shRNA portion of the project was endlessly frustrating and was a challenge for months.

Unfortunately the migrations that we intended to run did not get run because of the setbacks that were encountered. This does not mean that we will be stopping our work on the project but only that it’s been hindered. Some migration studies were done as mentioned, but not under the final conditions that were proposed and so my hypothesis remains to be challenged. I would hope that because of the continuing research in the lab surrounding EBI2 that this work when complete will be published in connection with additional research, but if not I will submit it for next year’s Utah and national conferences for undergraduate research.

I want to thank those in the lab that worked with me on this project namely: Dr.Poole, Daniel Clark (graduate student), Lissi Argueta, and Jordan Maybe. Also those outside our lab that helped give advice, time, or have committed in the future to do so namely: Dr. Wilson, Dr. Burnett, and Dr. McCleary.