After the project start date, I got started right away with necessary trainings for project completion. I received training for flow cytometry, microscopy, and mouse handling from our core facilities. I was trained by fellow lab members in relevant laboratory techniques. These involved generating cultures from primary cortical neurons and splenocytes. I also learned how to generate panels for immune cell staining for relevant immune profiling. I also optimized the co-culturing experiments I performed, by enriching for NKT cells and finding the best incubation periods, cell numbers, and reagent concentrations. I also learned how to work with mice at many developmental stages, including new-born pups. I learned how to extract the spleen, brain, skull bone marrow, dura, and choroid plexus from these mice at various developmental stages, and how to maximize the immune cell detection for each immune niche.
I have also performed immunofluorescent staining of mouse brain sections to pinpoint the choroid plexus and have stained for immature neuronal markers and CD1d presence. I have learned how to perform whole-mount staining of the choroid plexus, in order to visualize the structural integrity and the immune milieu in situ.
I have also analyzed a number of public and in-house databases to localize CD1d expression in the brain and distinguish the role of CD1d in relevant cells, including neurons and choroid plexus epithelium. Collecting data from multiple databases from our lab and from other labs guarantees that my findings are specific and reproducible.
I have generated a number of microscopic images, .fcs files, and code, all of which will be uploaded to public repositories. I have optimized tissue collection and immune cell purification for future single-cell experiments which will be made publicly available.
Furthermore, I successfully co-cultured the primary cortical neurons and NKT cells, and stained for activation markers, showing that these CD1d-expressing neurons can indeed activate NKT cells. This is the first time that such a neuroimmune communication has been discovered and researched. All of my results will be shared via poster presentations and talks, both internally and externally. I also plan on publishing my results in an open-access journal and in bioRxiv.
