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C. Pitsillides, P. Zamiri, J. Novak, C.P. Lin; Monitoring of Circulating Leukocytes by in vivo Flow Cytometry . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3589.
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Purpose: To visualize and monitor the immune system response during inflammatory and infective eye diseases and following ocular transplantation, by developing fluorescent probes to track circulating leukocytes in real time, in a living animal, using non–invasive optical methods. Methods: Injection of fluorescent antibodies into the circulation can interfere with the functions of targeted blood cells and can lead to depletion by the immune system, primarily via Fc–mediated mechanisms. In this study circulating CD4+ T cells were targeted by injecting mice with Cy5–conjugated IgG antibody fragments (anti–CD4 Fab and F(ab)2). Fluorescent cells were monitored at different time points, in the same animal, by in vivo confocal microscopy and flow cytometry. Circulation kinetics of fragment–labeled cells were compared to those of antibody–labeled cells, cells incubated with membrane specific dyes (DiD) or GFP–transfected cells. Results: Labeling circulating CD4+ T cells with fluorescent Fab or F(ab)2 antibody fragments, rather than the corresponding whole IgG molecule, results in significantly longer circulation times of the targeted cells, tracked continuously over a 24 hour period and up to several days. Conclusions: Developing probes to target circulating cells while at the same time imposing minimal disruption to the function of targeted cells will allow for extended monitoring of leukocytes in vivo. At the same time, the ability to selectively label specific subpopulations of cells will facilitate a better understanding of the mechanisms by which the immune system carries out its functions.
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