Flow cytometry,with double staining with Nile red and anti-CD3 antibody,to detect phospholipidosis in peripheral blood lymphocytes of rats treated with amiodarone |
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Authors: | Maria Elisa Perico Federica Crivellente Ivo Faustinelli Anna Suozzi Patrizia Cristofori |
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Institution: | (1) Department of Pathology, Safety Assessment, GlaxoSmithKline, Via Fleming 4, 37135 Verona, Italy |
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Abstract: | A flow cytometry method, to monitor peripheral lymphocytes phospholipidosis, has been set up using a single staining with
Nile red and double staining with Nile red and anti-CD3 monoclonal antibody. Blood has been collected from rats treated with
amiodarone (phospholipidogenic antiarrhythmic drug). By flow cytometer, it is possible to detect phospholipids, using Nile
red, a probe for intracellular lipids staining, changing its fluorescence on the stained lipid basis. CD3 antigen has been
selected to focus on T cells, to evaluate whether these cells are the target of phospholipidosis amiodarone-dependent. In
the study A, Sprague–Dawley rats were treated with three different doses (75, 150, and 300 mg kg−1 day−1) of amiodarone or vehicle alone, for 14 days, followed by 14 days of recovery: Data obtained show that by flow cytometry,
with Nile red alone, it is possible to detect a dose- and time-related response of phospholipidosis-positive lymphocytes;
a partial recovery is also assessed. In the study B, Sprague–Dawley rats were treated with a single dose (300 mg kg−1 day−1) of amiodarone, for 14 days: Data obtained show that animals treated with amiodarone have a significant increase of phospholipidosis-positive
lymphocytes (p = 0.008), in particular of CD3+ cells (p = 0.0056). Transmission electron microscopy analysis confirmed data obtained by flow cytometry. This work shows that flow
cytometry with Nile red could be a good tool to monitor ex vivo phospholipidosis in lymphocyte cells of animals treated with
amiodarone: The phospholipidogenic effect is more evident focusing on CD3+ T lymphocytes, thus suggesting that these cells are probably the target of phospholipidosis. |
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