Flow cytometry, with double staining with Nile red and anti-CD3 antibody, to detect phospholipidosis in peripheral blood lymphocytes of rats treated with amiodarone.
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.
Perico
Maria Elisa
ME
Department of Pathology, Safety Assessment, GlaxoSmithKline, Via Fleming 4, 37135, Verona, Italy. maria-elisa.e.perico@gsk.com
Crivellente
Federica
F
Faustinelli
Ivo
I
Suozzi
Anna
A
Cristofori
Patrizia
P
D000638:Amiodarone / Q000494:pharmacology; D000818:Animals; D000889:Anti-Arrhythmia Agents / Q000494:pharmacology; D000911:Antibodies, Monoclonal; D017252:CD3 Complex / Q000276:immunology*; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry / Q000379:methods*; D050356:Lipid Metabolism / Q000187:drug effects; D010078:Oxazines; D010743:Phospholipids / Q000032:analysis / Q000378:metabolism*; D051381:Rats; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D013997:Time Factors
D000889:Anti-Arrhythmia Agents; D000911:Antibodies, Monoclonal; D017252:CD3 Complex; D010078:Oxazines; D010743:Phospholipids; D000638:Amiodarone; C044808:nile red