Optimization of Fluorescent Phagocytic Assay Using Apoptotic Cell

Cedarville University DigitalCommons@Cedarville The Research and Scholarship Symposium The 2016 Symposium Apr 20th, 11:00 AM - 2:00 PM Optimization of Fluorescent Phagocytic Assay Using Apoptotic Cells Jacob L Brown Cedarville University, jacoblbrown@cedarville.edu Jason R Leigh Cedarville University, jasonleigh@cedarville.edu Ryan Marquardt Cedarville University, rmarquardt@cedarville.edu Cambria R Puffenberger Cedarville University, crpuffenberger@cedarville.edu Daniel J Stank Cedarville University, dstank@cedarville.edu See next page for additional authors Follow this and additional works at: http://digitalcommons.cedarville.edu/ research_scholarship_symposium Part of the Biology Commons, and the Immunopathology Commons Brown, Jacob L.; Leigh, Jason R.; Marquardt, Ryan; Puffenberger, Cambria R.; Stank, Daniel J.; and Pauley, Kaleb M., "Optimization of Fluorescent Phagocytic Assay Using Apoptotic Cells" (2016) The Research and Scholarship Symposium 45 http://digitalcommons.cedarville.edu/research_scholarship_symposium/2016/poster_presentations/45 This Poster is brought to you for free and open access by DigitalCommons@Cedarville, a service of the Centennial Library It has been accepted for inclusion in The Research and Scholarship Symposium by an authorized administrator of DigitalCommons@Cedarville For more information, please contact digitalcommons@cedarville.edu Presenters Jacob L Brown, Jason R Leigh, Ryan Marquardt, Cambria R Puffenberger, Daniel J Stank, and Kaleb M Pauley This poster is available at DigitalCommons@Cedarville: http://digitalcommons.cedarville.edu/research_scholarship_symposium/ 2016/poster_presentations/45 Optimization of Fluorescent Phagocytic Assay Using Apoptotic Cells Sjögren’s Syndrome is a systemic autoimmune disease that primarily affects the exocrine glands and is characterized by severe dry eyes and mouth Previous studies have shown that there are elevated levels of the microRNA miR-146a in Sjögren’s patients Mir-146a is a microRNA that has been found to be involved in down regulating inflammation Yet, in patients with Sjögren’s Syndrome, there is a large upregulation of miR-146a that exists alongside chronic inflammation This led us to investigate the role of miR-146a in Sjögren’s Syndrome We found that miR-146a upregulates phagocytosis of E coli by human macrophages Therefore, we hypothesized that this upregulation of phagocytosis should also apply to apoptotic cells In order to test this hypothesis, we had to optimize the induction of apoptosis in Jurkat cells and fluorescently label them for the phagocytosis assay We induced apoptosis using a topoisomerase inhibitor etoposide and performed a dose response curve to determine the optimal etoposide concentration We then assessed Jurkat viability using trypan blue exclusion and Annexin V staining We then fluorescently labeled the apoptotic cells with phrodo staining Phrodo is a pH-sensitive fluorophore that only fluoresces in acidic pH Finally we co-incubated the fluorescently labeled apoptotic jurkats with human macrophages (THP-1 cells) at a 4:1 ratio for the phagocytosis assay We tested 10 to 100 micromolar concentrations of etoposide and found the 40 micromolar concentration yielded optimal levels of apoptosis in the Jurkat cells The phrodo staining procedure was developed to fluorescently label the apoptotic cells Lastly, we performed the phagocytosis assay by incubating the fluorescently labeled apoptotic jurkat cells with THP-1 human macrophages which resulted in phagocytosis of the apoptotic Jurkats The conditions for the assay were optimized, and we plan to continue further research on miR-146a to investigate its effect on the phagocytosis of apoptotic cells  ... http://digitalcommons.cedarville.edu/research_scholarship_symposium/ 2016/poster_presentations/45 Optimization of Fluorescent Phagocytic Assay Using Apoptotic Cells Sjögren’s Syndrome is a systemic autoimmune disease that primarily... Jurkat cells The phrodo staining procedure was developed to fluorescently label the apoptotic cells Lastly, we performed the phagocytosis assay by incubating the fluorescently labeled apoptotic. .. that this upregulation of phagocytosis should also apply to apoptotic cells In order to test this hypothesis, we had to optimize the induction of apoptosis in Jurkat cells and fluorescently label