High‐Throughput Live Cell Imaging of Apoptosis

J.C. Puigvert1, Hans de Bont2, Bob van de Water2, Erik H.J. Danen2

1 Netherlands Toxicogenomics Centre, Leiden, The Netherlands, 2 Leiden University, Leiden, The Netherlands
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 18.10
DOI:  10.1002/0471143030.cb1810s47
Online Posting Date:  June, 2010
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Apoptosis is important for embryonic development, tissue homeostasis, and removal of cells with (potentially transforming) DNA lesions or other types of injuries. Functional genomics screens performed to unravel apoptotic signaling cascades in the context of toxicant‐induced cell injury commonly use apoptosis as an end‐point. Here, a method to detect the accumulation of apoptotic cells in real time that is well suited for high‐throughput screens is described. The method uses automated microscopy in a 96‐well format setting to visualize binding of fluorescent annexin V to the outer membrane leaflet of apoptotic cells. The automated image acquisition is followed by quantitative analysis using bioinformatics software. A protocol for each of the steps in this kinetic method is described, which includes the caspase‐dependent apoptotic response to toxic compounds in multiple cell types and demonstrates that RNAi‐based gene silencing of candidate apoptotic regulators affects the apoptosis kinetics as expected. This protocol will be useful for functional genomics as well as chemical (drug) screens. Curr. Protoc. Cell Biol. 47:18.10.1‐18.10.13. © 2010 by John Wiley & Sons, Inc.

Keywords: apoptosis; image analysis; annexin V; cell death; microscopy

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Table of Contents

  • Introduction
  • Basic Protocol 1: High‐Throughput Analysis of Apoptosis
  • Support Protocol 1: Preparation of Fluorescently Labeled Annexin V
  • Support Protocol 2: Quantitative Image Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: High‐Throughput Analysis of Apoptosis

  • Cells
  • 0.1% (w/v) gelatin in water
  • 1 mg/ml bovine plasma fibronectin solution (Sigma)
  • Phosphate buffered saline (PBS; appendix 2A)
  • 0.05% (w/v) trypsin/EDTA
  • Pre‐warmed culture medium
  • 0.4% trypan blue in PBS
  • Labeled annexin V (0.5 mg/ml stock concentration; see protocol 2)
  • Toxicant
  • Drugs to be tested
  • 100 mM z‐VAD‐fmk stock solution (Bachem)
  • 96‐ or 24‐well plates, e.g., CELLSTAR SensoPlate or CELLSTAR µClear plates (Greiner)
  • Hemacytometer
  • Inverted fluorescence microscope system with automated stage, automated focus control, and temperature‐ and CO 2‐controlled incubation system

Support Protocol 1: Preparation of Fluorescently Labeled Annexin V

  • Bacteria: E. coli BL21‐pLysS transformed with a pET/annexin V expression vector (available from Erik H.J. Danen; e.danen@lacdr.leidenuniv.nl)
  • LB medium containing 25 µg/ml chloramphenicol and 50 µg/ml kanamycin
  • IPTG
  • Spheroblast buffer (SBB; see recipe)
  • 10 mg/ml lysozyme in SBB
  • Ultracentrifugation working solution (UCB; see recipe)
  • Bovine brain extract (Sigma)
  • 1:2 (v/v) chloroform/methanol
  • Argon source
  • Diethyl ether
  • Liposome buffer (see recipe)
  • 300 mM CaCl 2
  • Buffer 1: liposome buffer (see recipe) plus 5 mM CaCl 2
  • Buffer 2: liposome buffer (see recipe) plus 10 mM EDTA
  • Buffer 3: 20 mM bis‐Tris, pH 6/0.02% (w/v) sodium azide
  • DEAE‐Sepharose fast‐flow column (∼14 × 150–mm)
  • 500 mM NaCl
  • Alexa fluor 488 protein labeling kit (Molecular Probes)
  • 37°C incubator
  • 250‐ml Erlenmeyer flasks
  • Spectrophotometer
  • 500‐ml centrifuge tubes
  • Refrigerated centrifuge
  • 50‐ml plastic tubes
  • 30‐ml ultracentrifugation tubes
  • Refrigerated ultracentrifuge
  • 250‐ml glass beakers
  • 10‐kDa dialysis membranes
  • Additional reagents and equipment for SDS PAGE gel electrophoresis (unit 6.1) and Coomassie staining (unit 6.6)

Support Protocol 2: Quantitative Image Analysis

  • Computer with tif images
  • Image Pro Plus software (Media Cybernetics; http://www.toxicologyleiden.nl)
  • Spreadsheet software (Excel)
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Literature Cited

Literature Cited
   Elmore, S. 2007. Apoptosis: A review of programmed cell death. Toxicol. Pathol. 35:495‐516.
   Gerke, V. and Moss, S.E. 2002. Annexins: From structure to function. Physiol. Rev. 82:331‐371.
   Hanshaw, R.G. and Smith, B.D. 2005. New reagents for phosphatidylserine recognition and detection of apoptosis. Bioorg. Medicinal Chem. 13:5035‐5042.
   Martin, S.J., Reutelingsperger, C.P.M., McGahon, A.J., Rader, J.A., van Schie, R.C.A.A., LaFace, D.M., and Green, D.R. 1995. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: Inhibition by overexpression of Bcl‐2 and Abl. J. Exp. Med. 182:1545‐1556.
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