Tanya Bogoslovsky1,2, Dragan Maric3, Yunhua Gong1, Baoxi Qu1, Kelly Yang4, Maria Spatz2, John Hallenbeck2, Ramon Diaz-Arrastia1. 1. Center for Neuroscience & Regenerative Medicine, Uniformed Services University of Health Sciences, 12725 Twinbrook Pkwy, Rockville, MD 20852, USA. 2. National Institute of Neurological Disorders and Stroke, Stroke Branch, 10 Center Drive, Bethesda, MD 20814, USA. 3. National Institute of Neurological Disorders and Stroke, Flow Cytometry Core Facility, 49 Convent Drive, Bethesda, MD 20814, USA. 4. National Institute of Neurological Disorders and Stroke, 10 Center Drive, Bethesda, MD 20814, USA.
Abstract
AIMS: Endothelial progenitor cells (EPCs) are markers of vascular repair. Increased numbers of circulating endothelial cells (ECs) are associated with endothelial damage. MATERIALS & METHODS: We enumerated EPC-EC by using Enrichment kit with addition of anti-human CD146-PE/Cy7 from peripheral blood mononuclear cell (PBMC) isolated either by red blood cell (RBC) lysing solution or by Ficoll centrifugation, and from fresh and preserved samples. PBMCs were quantified by flow cytometry. RESULTS: RBC lysis yielded higher percentage of PBMC (p = 0.0242) and higher numbers of PBMC/ml (p = 0.0039) than Ficoll. Absolute numbers of CD34(+)CD133(+)VEGFR2(+) and CD146(+)CD34(+)VEGFR2(+) were higher (p = 0.0117 for both), when isolated by RBC lysis than by Ficoll, when no difference in other subsets was found. Cryopreservation at -160°C and -80°C and short-term preservation at room temperature decreased EPC-EC. CONCLUSIONS: Our data support use of fresh samples and isolation of PBMC from human blood by RBC lysis for enumeration of EPC and EC.
AIMS: Endothelial progenitor cells (EPCs) are markers of vascular repair. Increased numbers of circulating endothelial cells (ECs) are associated with endothelial damage. MATERIALS & METHODS: We enumerated EPC-EC by using Enrichment kit with addition of anti-human CD146-PE/Cy7 from peripheral blood mononuclear cell (PBMC) isolated either by red blood cell (RBC) lysing solution or by Ficoll centrifugation, and from fresh and preserved samples. PBMCs were quantified by flow cytometry. RESULTS: RBC lysis yielded higher percentage of PBMC (p = 0.0242) and higher numbers of PBMC/ml (p = 0.0039) than Ficoll. Absolute numbers of CD34(+)CD133(+)VEGFR2(+) and CD146(+)CD34(+)VEGFR2(+) were higher (p = 0.0117 for both), when isolated by RBC lysis than by Ficoll, when no difference in other subsets was found. Cryopreservation at -160°C and -80°C and short-term preservation at room temperature decreased EPC-EC. CONCLUSIONS: Our data support use of fresh samples and isolation of PBMC from human blood by RBC lysis for enumeration of EPC and EC.