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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Abstract

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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Materials

Basic Protocol 1: High‐Resolution Image‐Cytometry Multivariate Analysis of EdU‐Labeled Cells

  Materials
  • 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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Figures

Videos

Literature Cited

  Basiji, D.A., Ortyn, W.E., Liang, L., Venkatachalam, V., and Morrissey, P. 2007. Cellular image analysis and imaging by flow cytometry. Clinics Lab. Med. 27:653‐670.
  Bernaś, T., Berniak, K., Rybak, P., Zarębski, M., Zhao, H., Darzynkiewicz, Z., and Dobrucki, J.W. 2013. Analysis of spatial correlations between patterns of DNA damage response and DNA replication in nuclei of cells subjected to replication stress or oxidative damage. Cytometry A 83:825‐932.
  Berniak, K., Rybak, P., Bernaś, T., Zarębski, M., Biela, E., Zhao, H., Darzynkiewicz, Z., and Dobrucki, J.W. 2013. Relationship between DNA damage response, initiated by camptothecin or oxidative stress, and DNA replication, analyzed by quantitative 3D image analysis. Cytometry A 83:913‐824.
  Buck, S.B., Bradford, J., Gee, K.R., Agnew, B.J., Clarke, S.T., and Salic, A. 2008. Detection of S‐phase cell cycle progression using 5‐ethynyl‐2'‐deoxyuridine incorporation with click chemistry, an alternative to using 5‐bromo‐2'‐deoxyuridine antibodies. BioTechniques 44:927‐929.
  Chattopadhyay, P.K., Hogerkorp, C.M., and Roederer, M. 2008. A chromatic explosion: The development and future of multiparameter flow cytometry. Immunology 125:441‐449.
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  Darzynkiewicz, Z., Traganos, F., and Melamed M.R. 1980. New cell cycle compartments identified by multiparameter flow cytometry. Cytometry 1:98‐108.
  Darzynkiewicz, Z., Zhao, H., Halicka, H.D., and Li, J. 2011a. Cytometry of DNA replication and RNA synthesis: Historical perspective and recent advances based on “Click chemistry”. Cytometry A 79:328‐337.
  Darzynkiewicz, Z., Traganos, F., Zhao, H., Halicka, H.D., Skommer, J., and Wlodkowic, D. 2011b. Analysis of individual molecular events of DNA damage response by flow‐ and image‐assisted cytometry. Methods Cell Biol. 103:115‐147.
  Furia, L., Pelicci, P.G., and Faretta, M. 2013a. A computational platform for robotized fluorescence microscopy (I): High‐content image‐based cell‐cycle analysis. Cytometry A 83:333‐343.
  Furia, L., Pelicci, P.G., and Faretta, M. 2013b. A computational platform for robotized fluorescence microscopy (II): DNA damage, replication, checkpoint activation, and cell cycle progression by high‐content high‐resolution multiparameter image‐cytometry. Cytometry A 83:344‐355.
  Hell, S.W. 2007. Far‐field optical nanoscopy. Science 316:1153‐1158.
  Kamentsky, L.A. 2001. Laser scanning cytometry. Methods Cell Biol. 63:51‐87.
  Li, X., Melamed, M.R., and Darzynkiewicz, Z. 1996. Detection of apoptosis and DNA replication by differential labeling of DNA strand breaks with fluorochromes of different color. Exp. Cell Res. 222:28‐37.
  Loo, L.H., Wu, L.F., and Altschuler, S.J. 2007. Image‐based multivariate profiling of drug responses from single cells. Nat. Methods 4:445‐453.
  Mach, W.J., Thimmesch, A.R., Orr, J.A., Slusser, J.G., and Pierce, J.D. 2010. Flow cytometry and laser scanning cytometry, a comparison of techniques. J. Clin. Monitor. Comput. 24:251‐259.
  McCoy, J.P. Jr. 2011. High‐content screening: Getting more from less. Nat. Methods 8:390‐391.
  Neumann, B., Walter, T., Heriche, J.K., Bulkescher, J., Erfle, H., Conrad, C., Rogers, P., Poser, I., Held, M., Liebel, U., Cetin, C., Sieckmann, F., Pau, G., Kabbe, R., Wunsche, A., Satagopam, V., Schmitz, M.H., Chapuis, C., Gerlich, D.W., Schneider, R., Eils, R., Huber, W., Peters, J.M., Hyman, A.A., Durbin, R., Pepperkok, R., and Ellenberg, J. 2010. Phenotypic profiling of the human genome by time‐lapse microscopy reveals cell division genes. Nature 464:721‐727.
  Ortyn, W.E., Perry, D.J., Venkatachalam, V., Liang, L., Hall, B.E., Frost, K., and Basiji, D.A. 2007. Extended depth of field imaging for high speed cell analysis. Cytometry A 71:215‐231.
  Pawley, J.B. 1995. Handbook of Biological Confocal Microscopy. Plenum, New York.
  Salic, A. and Mitchison, T.J. 2008. A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc. Natl. Acad. Sci. U.S.A. 105:2415‐2420.
Internet Resources
  http://en.wikipedia.org/wiki/High‐content_screening
  A very basic and general introductory site with useful links and references.
  http://www.cellprofiler.org/
  A free powerful tool to perform image analysis in cell biology.
  http://www.micro‐manager.org/
  An open source microscopy software.
  http://imagej.nih.gov/ij/
  The site of the ImageJ java‐based image analysis platform.
  http://fiji.sc/Fiji
  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.
  http://www.cyto.purdue.edu/Purdue_software
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