Over the past two weeks, I have performed pilot experiments to finalize the design of my final project. One of these pilots compared an infected wild type model with an uninfected one; wild type means that the model has not been genetically modified in any way.
A day after infection, I examined spleen for the presence of certain dendritic cell variants. Dendritic cells are one of many types of white blood cells, and some of their subsets are found in greater numbers during infection. Therefore, I expected to find more of these dendritic cells after lymphocytic choriomeningitis virus (LCMV) infection. Dendritic cells are an essential part of the immune system, as they detect foreign bodies, such as pathogens, and present the pathogen’s antigens to T cells. Antigens are the surface proteins on cells that act as a recognition system. Your body has a set of antigens that are unique, so your immune cells can recognize you and find foreign cells. The two types of dendritic cells I was interested in, conventional and plasmacytoid, both produce various cell-surface activation markers in response to infection. I used this as a measure of immune response to the virus.
On the whole, my experiment was successful! Using flow cytometry, I analyzed cells isolated from spleen to find dendritic cells. Flow cytometry is a method used to detect proteins on the surface of cells by adding antibodies, attached to a fluorescent molecule, to a mixture of cells. The flow cytometer machine takes these cells up through a nozzle and lines them up single file. Then, one by one, they pass in front of a laser. Inside the machine, there are photodetectors that can sense the colors on the cells. Because each antibody will bind to only one kind of protein on the cell surface, and the color is linked to the antibody, we can see what is on the surface of each cell that passes through the machine. For a more in-depth discussion of flow cytometry, I recommend the Wikipedia article (http://en.wikipedia.org/wiki/Flow_cytometry). Also, there’s also a good interactive description by Oregon State University (http://www.unsolvedmysteries.oregonstate.edu/flow_06).
My infected dendritic cells displayed more activation markers than my uninfected cells, which is the expected, healthy response. The purpose of this pilot was to assess my experiment’s design – part of the difficulty of flow cytometry is choosing the right antibodies so we can detect the right kinds of cells in addition to the proteins of interest. And, since each antibody has to be associated with a different color flurochrome so we can detect them all separately, the color panel needs to be designed carefully to avoid any overlap in color spectrum.
With the positive result of this experiment, I will move onto a model more similar to a human: this time I will be considering whether the transgenic model with the human, normal version of PTPN22 can rescue the immune response of a knockout model – this latter model completely lacks PTPN22, and so does not mount an immune response. If the normal human version, known as LypR, works, I will be able to consider the human, mutated, SLE-associated version called LypW in a future infection experiment.
The past two weeks have been the culmination of a month’s research in the technical background of flow cytometry, and I’m very pleased to be mounting experiments independently to get strong data and results. I now feel like a real scientist, working into the night and being excited about it!