Confocal Microscopy for High‐Resolution and High‐Content Analysis of the Cell Cycle

Laura Furia1, Piergiuseppe Pelicci1, Mario Faretta1

1 Department of Experimental Oncology, European Institute of Oncology, Milan
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.42
DOI:  10.1002/0471142956.cy0742s70
Online Posting Date:  October, 2014
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Abstract

Optical fluorescence microscopy offers a wide range of technological solutions to address many questions in biomedical research. Spatial resolution has been greatly improved by the use of confocal microscopes, providing a 3‐D analysis of the intracellular space. Automation has contributed to make confocal analysis available for high‐content image cytometry studies. However, the storage, browsing, and analysis of the amount of data generated can challenge the feasibility of such studies. Presented in this chapter is a multistep acquisition and analysis protocol that can bypass such difficulties by an analysis‐driven data collection. Cell‐cycle analysis of low‐resolution data can be employed to select cell populations of interest that can then be imaged at extremely high resolution and subjected to high‐content analysis. Curr. Protoc. Cytom. 70:7.42.1‐7.42.14. © 2014 by John Wiley & Sons, Inc.

Keywords: image cytometry; cell cycle; S phase; high content; high resolution; automated microscopy; confocal microscopy

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: High‐Resolution Confocal Mutivariate Analysis of EdU‐Labeled Cells
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: High‐Resolution Confocal Mutivariate Analysis of EdU‐Labeled Cells

  Materials
  • Cells of interest (also see unit 7.41)
  • Chromomycin A3 (Sigma‐Aldrich)
  • Phosphate‐buffered saline (PBS; appendix 2A) containing 70 mM MgCl 2
  • Non‐hardening mounting medium with RI close to 1.52: e.g., ProLong (Life Technologies)
  • Clear nail polish
  • Additional reagents and equipment for cell culture and EdU immunostaining (unit 7.41)
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Figures

Videos

Literature Cited

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Internet Resources
  http://www.leica‐microsystems.com/products/confocal‐microscopes/details/product/leica‐hcs‐a‐2/
  Link to the system employed in this work for data collection.
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