High‐Resolution Cytometry for High‐Content Cell Cycle Analysis

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 7.41
DOI:  10.1002/0471142956.cy0741s70
Online Posting Date:  October, 2014
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One of the major limitations of flow cytometry (FCM) is the absence of an intracellular view. Automated microscopy and image analysis, together with technological developments, led to new approaches in cytometry that bypass the above limitation, introducing high resolution, high content, and large statistical sampling. However, few attempts have been made, until now, to translate the wide repertoire of FCM assays into high‐content image screening. This unit describes the implementation of an acquisition and analysis protocol for evaluation of the cell cycle by automated microscopy. The approach grants the possibility to perform simultaneous analysis of a high number of different parameters. A large part of this unit is devoted to the description of hardware features that can optimize the recorded information together with the acquisition and analysis procedures employed to produce good‐quality data. Curr. Protoc. Cytom. 70:7.41.1‐7.41.15. © 2014 by John Wiley & Sons, Inc.

Keywords: image cytometry; cell cycle; S phase; high‐content analysis; high resolution; automated microscopy; wide‐field microscopy

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Table of Contents

  • Introduction
  • Basic Protocol 1: High‐Resolution Image‐Cytometry Multivariate Analysis of EdU‐Labeled Cells
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: High‐Resolution Image‐Cytometry Multivariate Analysis of EdU‐Labeled Cells

  • Mammary epithelial, non‐transformed cell line MCF10A
  • 0.5% porcine gelatin (Sigma‐Aldrich)
  • Phosphate‐buffered saline (PBS)
  • MCF10A cell culture medium: DMEM/Ham's F12 medium (1:1)/5% fetal bovine serum/2 mM glutamine
  • 5‐Ethyny‐l‐2′‐deoxyuridine (EdU) (Life Technologies)
  • 4% paraformaldheyde
  • 0.1% Triton X‐100 in PBS
  • Click‐iT EdU Imaging kit (Life Technologies)
  • 5% BSA
  • Primary antibodies
  • Secondary antibodies
  • Mouse immunoglobulin (Jackson ImmunoResearch)
  • Directly conjugated monoclonal antibodies
  • 10 μg/ml DAPI in PBS
  • Mowiol or other fluorescence microscopy mounting medium (Merck‐Millipore)
  • 2‐cm diameter, 0.17‐mm thick glass coverslips
  • Automated microscope with image acquisition hardware and software
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Literature Cited

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Internet Resources
  A very basic and general introductory site with useful links and references.
  A free powerful tool to perform image analysis in cell biology.
  An open source microscopy software.
  The site of the ImageJ java‐based image analysis platform.
  Some useful free FCM software packages.
  Another image processing package derived from ImageJ.
  For black‐box screening instrumentation refer to the Web sites of major microscope manufacturers.
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