EXTENDING A FLOW CYTOMETRIC ASSAY TO ASSESS RICKETTSIA PARKERI INFECTIVITY IN SKIN-INFILTRATING LEUKOCYTES
Yuan, Sean
2023
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Rickettsia parkeri is a tick-borne obligate intracellular pathogen in the spotted fever groupRickettsia spp. (SFGR) that causes spotted fever rickettsiosis (SFR). Our lab studies SFR using the guinea pig model and R. parkeri transmission by its primary vector, the Gulf coast tick. Here, we expand a previously developed flow cytometric (FCM) assay for immunophenotyping cellular infiltrates in guinea ... read morepig skin to identify host cells targeted by R. parkeri. Specifically, we are testing an anti-SFGR antibody designed for immunohistochemistry (IHC) in an in vitro FCM experiment using differentiated THP-1 cells (human leukemia monocytic cell line) prior to moving to an animal study. First, we determined the number of THP-1 cells required to seed a 6-well plate to recover 1 x 10 6 viable differentiated cells per well. Then, we confirmed the percentage of formaldehyde required to inactivate R. parkeri when fixing cells in the FCM protocol. Next, we are determining the minimum time necessary for R. parkeri (MOI of 10) to invade host cells. We will apply those data to identify the multiplicity of infection (MOI) required to infect approximately 20 percent of the host cells for testing our FCM assay. We labeled the anti-SFGR antibody with the fluorophore CF405L in-house and are performing a titration to tell us if the antibody would be useful for flow cytometry and, if so, at what optimal dilution. We anticipate expanding our FCM assay to detect infected leukocytes in spiked guinea pig blood before moving to in vivo studies. Ultimately, this work will allow us to better understand disease progression and pathogenesis during infection after natural tick transmission in the guinea pig model.
Cummings School of Veterinary Medicine Student Summer Research 2023.read less - ID:
- w6634j24x
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