Identification of Endothelial Cells and Progenitor Cell Subsets in Human Peripheral Blood

Myka L. Estes1, Julie A. Mund1, David A. Ingram2, Jamie Case1

1 Herman B. Wells Center for Pediatric Research, Indianapolis, Indiana, 2 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.33
DOI:  10.1002/0471142956.cy0933s52
Online Posting Date:  April, 2010
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An assay for circulating cell subsets in human peripheral blood by flow cytometry is used as a biomarker to determine cardiovascular disease risk and tumor responsiveness to chemotherapy since endothelial progenitor cells (EPCs) function in vasculogenesis and angiogenesis. Despite analytical advances in polychromatic flow cytometry (PFC), conventional approaches are routinely utilized to enumerate and isolate EPCs, which has led to varied results in clinical studies, potential cellular misidentification, and thus a lack of a plausible biological explanation for how purported EPCs function. Herein, a reproducible PFC protocol is provided to identify a rare circulating endothelial colony‐forming cell (ECFC) with proliferative potential, along with a population of circulating progenitor cells (CPCs) in which the ratio analysis distinguishes between healthy and disease populations. In sum, a reliable PFC protocol, which can be used to investigate the roles of human hematopoietic and endothelial elements in the growth and maintenance of the vasculature, is described. Curr. Protoc. Cytom. 52:9.33.1‐9.33.11. © 2010 by John Wiley & Sons, Inc.

Keywords: endothelial cells; circulating endothelial cells; endothelial progenitor cells; circulating endothelial progenitors; human peripheral blood

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Identification of Endothelial Colony Forming Cells and Circulating Progenitor Cell Subsets in Human Peripheral Blood by Polychromatic Flow Cytometry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Identification of Endothelial Colony Forming Cells and Circulating Progenitor Cell Subsets in Human Peripheral Blood by Polychromatic Flow Cytometry

  • Peripheral blood (PB)
  • EDTA collection tubes (Vacutainer; BD, cat. no. 366450)
  • Phosphate buffered saline (PBS; Invitrogen, cat. no. 20012‐027)
  • Ficoll‐Paque Plus (GE Healthcare, cat. no. 17‐1440‐03)
  • Fetal bovine serum (FBS; see recipe)
  • FcR‐blocking agent (Miltenyi Biotec, cat. no. 130‐059‐901)
  • CD34‐PE (BD Biosciences, cat. no. 550761)
  • AC133‐APC (Miltenyi Biotec, cat. no. 130‐090‐826)
  • CD14‐PE‐Cy5.5 (abCAM, cat. no. ab25395)
  • CD45‐APC‐AF750 (Invitrogen, cat. no. MHCD4527)
  • CD31‐FITC (BD Biosciences, cat. no. 555445)
  • CD235a (glyA; R&D Systems, cat. no. MAB1228)
  • Pacific blue monoclonal antibody labeling kit (Invitrogen, cat. no. P30013)
  • CD41a (Invitrogen, cat. no. MHCD4100)
  • LIVE/DEAD fixable violet dead cell stain kit (Invitrogen, cat. no. L34955)
  • FACS formaldehyde (see recipe), optional
  • BD CompBeads (BD Biosciences, cat. no. 552843)
  • Amine polymer microspheres (Bangs Lab, cat. no. PA04N)
  • Quantum simply cellular beads (Bangs Lab, cat. no. 816) or equivalent
  • 15‐ and 50‐ml conical centrifuge tubes (BD Falcon, cat. no. 352097 and 352098, respectively)
  • 10‐ and 30‐ml disposable syringes (BD Falcon, cat. no. 309604 and 309650, respectively)
  • Mixing cannulas (UNO Medical, cat. no. 500.11.012)
  • 14 × 5–ml round‐bottom polystyrene tubes (BD Falcon, cat. no. 352008)
  • Aluminum foil
  • 40‐µm cell strainers (BD Falcon, cat. no. 352340)
  • LSRII flow cytometer with 405‐nm, 488‐nm, and 633‐nm laser configurations (BD)
  • Flowjo software (Treestar)
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Literature Cited

Literature Cited
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