Characterization of Surface FAS—Quantitative Morphological Analysis Using Quantitative Imaging Cytometry

Heather Ireland Zecchini1, William R. English2, Stefanie Reichelt1

1 Light Microscopy Core Facility, Cancer Research UK Cambridge Research Institute, Cambridge, United Kingdom, 2 Department of Oncology, School of Medicine, University of Sheffield, Sheffield, United Kingdom
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.25
DOI:  10.1002/0471142956.cy1225s59
Online Posting Date:  January, 2012
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Abstract

Long‐term benefits of cardiovascular therapeutic interventions are often reduced due to reocclusion of the treated vessels. Therefore, a therapeutic strategy to reduce neointima formation would be of great interest. A better understanding of the mechanisms underlying apoptosis in vascular smooth muscle cells would be key in the development of such a strategy. Efficient activation of caspase‐8 regulated apoptosis is largely dependent on the aggregation of FAS at the cell surface, and a method to facilitate the study of FAS clustering and its role in apoptosis in these cells would be particularly useful. Here we describe a protocol for the antibody staining of cell surface FAS foci and the application of the iCys Research Imaging Cytometer for the quantitative and morphological analysis of the cell surface FAS foci. Curr. Protoc. Cytom. 59:12.25.1‐12.25.17. © 2012 by John Wiley & Sons, Inc.

Keywords: laser scanning cytometry; high‐content cell analysis; cell surface FAS foci; apoptosis DISC

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Staining of Human Aortic Vascular Smooth Muscle Cells for Quantitative Analysis
  • Support Protocol 1: Image Acquisition
  • Support Protocol 2: Image Analysis
  • Support Protocol 3: Data Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Staining of Human Aortic Vascular Smooth Muscle Cells for Quantitative Analysis

  Materials
  • Human aortic vascular smooth muscle cells (hVSMCs were purchased cryopreserved; PromoCell)
  • Cell culture medium (see recipe)
  • 0.05% trypsin/EDTA (Invitrogen)
  • Cell culture medium containing inhibitors in DMSO (e.g., receptor tyrosine kinase inhibitors to Met and PI3K; Merck Biochemicals, cat. nos. SU11274 and LY294002) or the vehicle control, optional
  • 4% w/v paraformaldehyde (PFA) fixative in Hank's buffered saline solution (HBSS) with Ca2+/Mg2+(Invitrogen)
  • Hank's buffered saline solution (HBSS) with Ca2+/Mg2+
  • Phosphate‐buffered saline (PBS; see recipe)
  • 100 mM glycine in PBS, pH 7.4
  • Mouse IgM monoclonal anti‐FAS clone CH‐11 (Millipore)
  • IgM Isotype control antibody (Sigma)
  • Goat serum (Sigma)
  • AlexaFluor‐488 anti‐mouse IgM (Invitrogen)
  • HCS CellMask Deep Red stain (Invitrogen, cat. no. H32721)
  • 37°C, 5% CO 2 incubator
  • IbiTreat µ‐slides (Ibidi, cat. no. 80606)

Support Protocol 1: Image Acquisition

  Materials
  • hVSMCs on µ‐slides (see the protocol 1)
  • iCys Research Imaging Cytometer (CompuCyte); see Introduction above for additional information
  • iCys software
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Figures

Videos

Literature Cited

Literature Cited
   Clarke, M. and Bennett, M. 2006. Defining the role of vascular smooth muscle cell apoptosis in atherosclerosis. Cell Cycle 5:2329‐2331.
   Darzynkiewicz, Z., Bedner, E., Li, X., Gorczyca, W., and Melamed, M.R. 1999. Laser‐scanning cytometry: A new instrumentation with many applications. Exp. Cell Res. 249:1‐12.
   Gloire, G., Charlier, E., and Piette, J. 2008. Regulation of CD95/APO‐1/Fas‐induced apoptosis by protein phosphatases. Biochem. Pharmacol. 76:1451‐1458.
   Henriksen, M., Miller, B., Newmark, J., Al‐Kofahi, Y., and Holden, E. 2011. Laser scanning cytometry and its applications: A pioneering technology in the field of quantitative imaging cytometry. Methods Cell Biol. 102:159‐205.
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   Mollinedo, F. and Gajate, C. 2006. Fas/CD95 death receptor and lipid rafts: New targets for apoptosis‐directed cancer therapy. Drug Resist. Updat. 9:51‐73.
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   Wang, X., DeFrances, M.C., Dai, Y., Pediaditakis, P., Johnson, C., Bell, A., Michalopoulos, G.K., and Zarnegar, R. 2002. A mechanism of cell survival: sequestration of Fas by the HGF receptor Met. Molecular Cell 9:411‐421.
   White, S.J. and Newby, A.C. 2002. Gene therapy for all aspects of vein‐graft disease. J. Card. Surg. 17:549‐555.
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