Multi‐Parametric Analysis of Cell Death Pathways Using Live‐Cell Microscopy

Gaurav N. Joshi1, David A. Knecht1

1 Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.40
DOI:  10.1002/0471140856.tx0440s58
Online Posting Date:  November, 2013
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Abstract

Programmed cell death is a complex process with new forms being discovered with regularity. Each pathway has a distinct and overlapping biochemical and physiological change occurring as the cell prepares for death. Detection of these changes can be facilitated by the availability of various fluorescent probes and advances in microscope systems. By analyzing these probes over time using fluorescence microscopy, the changes that occur in each cell en route to death can be analyzed on a cell‐by‐cell basis. While the timing of events varies considerably from cell to cell, it has been found that the sequence of events is highly conserved. Transient events, which would be difficult to detect using population averaging techniques, are readily detected when cells are analyzed individually in time lapse. The protocols in this unit describe using probes for real‐time imaging of one of the apoptotic cell death pathways using various inducers, as well as the associated hardware necessary for imaging so that the imaging itself is not affecting cell viability. Curr. Protoc. Toxicol. 58:4.40.1‐4.40.31. © 2013 by John Wiley & Sons, Inc.

Keywords: apoptosis; fluorescence microscopy; real‐time imaging; image analysis; FRET

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Plating of Cells
  • Basic Protocol 2: Environmental Chambers for Temperature Regulation
  • Basic Protocol 3: Detecting Lysosomal Leakage
  • Support Protocol 1: Quantification of an Increase in Nuclear Fluorescence
  • Basic Protocol 4: Evaluating Changes in Mitochondria Membrane Potential
  • Basic Protocol 5: Transfection of Cells
  • Basic Protocol 6: Bax‐Induced Mitochondria Outer Membrane Permeabilizaton (MOMP)
  • Basic Protocol 7: Real‐Time Detection of Caspase Activity
  • Support Protocol 2: Quantification and Generation of Pseudocolor Ratiometric Images from FRET Data
  • Basic Protocol 8: Detecting Phosphatidylserine Externalization
  • Basic Protocol 9: Detecting Alterations in Nuclear Morphology Using Hoechst 33342
  • Basic Protocol 10: Propidium Iodide Assay
  • Basic Protocol 11: Multi‐Parametric Analysis of Apoptosis
  • Basic Protocol 12: Inducing Lysosomal Leakage and Cell Death
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Plating of Cells

  Materials
  • MH‐S alveolar macrophages (ATCC# 2019)
  • RPMI complete medium (see recipe)
  • 37°C, 5% CO 2 incubator
  • Pipets
  • 35‐mm Bioptechs dishes (Fisher scientific, cat. no. 12‐071‐07), 35‐mm glass‐bottom petri dishes (WillCo wells, cat. no. GWSt‐3522 or Matek Corp.), or 8‐well chamber slide (Thermo Scientific Nunc Lab‐Tek II chambered cover glass, cat. no. 12565338)

Basic Protocol 2: Environmental Chambers for Temperature Regulation

  Materials
  • Cells
  • Appropriate cell dye(s) (see Basic Protocols protocol 33 to protocol 1210)
  • Cell death inducer (see protocol 14)
  • Mineral oil
  • 70% ethanol
  • L‐900 detergent (Nalgene)
  • Delta T dish system (Bioptechs)
  • Zeiss Axiovert 200 M widefield fluorescence microscope
  • Plexiglas full microscope enclosure
  • 8‐well chambered slide or WillCo dish
  • Bold‐line cage incubator (Okolab)
  • Live‐cell chamber (Pathology Devices)
  • Nikon A1R confocal microscope
  • Biological safety cabinet equipped with UV sterilizing lamp

Basic Protocol 3: Detecting Lysosomal Leakage

  Materials
  • FITC‐dextran (4‐kD) (Sigma) (100 mg/ml stock in water, store at −20°C)
  • RPMI complete medium (see recipe)
  • CO 2‐independent medium (Invitrogen)
  • Drug or particle (see protocol 14)
  • 37°C, 5% CO 2 incubator
  • 37°C ambient air incubator
  • Fluorescence microscope (widefield or confocal; widefield recommended) with image acquisition capabilities
  • 1.5‐ml microcentrifuge tubes
  • Vortexer
  • Additional reagents and equipment for plating cells (see protocol 1)

Support Protocol 1: Quantification of an Increase in Nuclear Fluorescence

  Materials
  • Data from FITC‐dextran and transmitted light or DIC channels (see protocol 3)
  • Image J (available at http://rsbweb.nih.gov/ij/download.html; Schneider et al., )
  • Microsoft Excel

Basic Protocol 4: Evaluating Changes in Mitochondria Membrane Potential

  Materials
  • 50 µM tetramethylrhodamine ester in DMSO (TMRE, Sigma)
  • RPMI complete medium (see recipe)
  • CO 2‐independent medium (Invitrogen)
  • Oligomycin (Sigma)
  • Carbonyl cyanide 4‐(trifluoromethoxy)​phenylhydrazone (FCCP, Sigma)
  • Drugs/compounds for testing (see protocol 14)
  • 37°C incubator
  • 37°C, 5% CO 2 incubator
  • 1.5‐ml microcentrifuge tubes
  • Fluorescence microscope with image acquisition hardware and software

Basic Protocol 5: Transfection of Cells

  Materials
  • Cells
  • DNA to be transfected (obtained by midi‐prep or maxi‐prep)
  • RPMI‐1640 incomplete medium (without serum and antibiotic)
  • Lipofectamine 2000 (Invitrogen) or Fugene HD (Promega)
  • RPMI‐1640 complete medium (see recipe)
  • Sterile 35‐mm glass‐bottom petri dishes (e.g., WillCo dish)
  • 37°C, 5% CO 2 incubator
  • Fluorescence microscope
  • 5‐ml sterile polystyrene tubes

Basic Protocol 6: Bax‐Induced Mitochondria Outer Membrane Permeabilizaton (MOMP)

  Materials
  • MH‐S cells
  • GFP‐hBax DNA (Addgene, cat. no. 19741)
  • Drugs/compounds for treatment (see protocol 14)
  • Confocal microscope equipped with a temperature and humidity controlled system
  • 60× oil‐immersion lens
  • 1.5‐ml microcentrifuge tubes
  • Additional reagents and equipment for transfection (see protocol 6)

Basic Protocol 7: Real‐Time Detection of Caspase Activity

  Materials
  • Effector caspase DNA (CFP‐DEVD‐YFP) (Addgene, cat. no. 24537)
  • Zeiss Axiovert 200 M wide‐field fluorescence microscope
  • Filter sets
  • CFP imaging (Chroma, cat. no. 31044v2)
    • CFP (ex): 436/20 nm
    • Beam splitter: 455 nm
    • CFP (em): 480/40 nm
  • YFP‐FRET imaging (Chroma, cat. no. 31052; cyan/topaz energy transfer)
    • CFP (ex): 436/20 nm
    • Beam splitter: 455 nm
    • YFP‐FRET (em): 535/30 nm
  • Additional reagents and equipment for plating MH‐S cells (see protocol 1) and FRET‐sensor (see protocol 6)

Support Protocol 2: Quantification and Generation of Pseudocolor Ratiometric Images from FRET Data

  Materials
  • Image J (available at http://rsbweb.nih.gov/ij/download.html; Schneider et al., )
  • Data from CFP and YFP‐FRET channels (see protocol 8)
  • Microsoft Excel

Basic Protocol 8: Detecting Phosphatidylserine Externalization

  Materials
  • AnnexinV‐FITC (BD Biosciences), store at 4°C
  • Desired drug or particle (see protocol 14)
  • Epifluorescence microscope (or confocal)
  • 1.5‐ml microcentrifuge tubes
  • Vortex
  • Additional reagents and equipment for plating cells in Bioptechs dishes (see protocol 1)

Basic Protocol 9: Detecting Alterations in Nuclear Morphology Using Hoechst 33342

  Materials
  • Hoechst 33342 (5 mg/ml stock in DMSO, Sigma), store at −20°C
  • RPMI complete medium (see recipe)
  • CO 2‐independent medium (Sigma)
  • Desired drug or particle (see protocol 14)
  • 1.5‐ml microcentrifuge tubes
  • 37°C, 5% CO 2 incubator
  • Microscope with appropriate filter sets
  • Additional reagents and equipment for cell plating (see protocol 1)

Basic Protocol 10: Propidium Iodide Assay

  Materials
  • RPMI complete medium (see recipe)
  • CO2‐independent medium (Sigma)
  • Propidium iodide (1 mg/ml stock in water, Sigma)
  • Desired drug or particle (see protocol 14)
  • Hydrogen peroxide (30% v/v, Fisher scientific)
  • 37°C incubator
  • Wide‐field fluorescence microscope
  • 1.5‐ml microcentrifuge tubes
  • Vortex
  • Additional reagents and equipment for plating cells (see protocol 1)

Basic Protocol 11: Multi‐Parametric Analysis of Apoptosis

  Materials
  • Staurosporine (1 mM stock in DMSO, Sigma), store at −20°C
  • L‐leucyl L‐leucine methyl ester (Leu‐Leu‐O‐Me, 500 mM stock in DMSO, Sigma), store at −20°C
  • 3‐µm spherical silica particle (Alltech Associates, Grace Davison)
  • 1.5‐ml microcentrifuge tubes
  • Heating oven
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Figures

Videos

Literature Cited

Literature Cited
  Albeck, J., Burke, J., Aldridge, B., Zhang, M., Lauffenburger, D., and Sorger, P. 2008. Quantitative analysis of pathways controlling extrinsic apoptosis in single cells. Mol. Cell 30:11‐25.
  Bidère, N., Lorenzo, H.K., Carmona, S., Laforge, M., Harper, F., Dumont, C., and Senik, A. 2003. Cathepsin D triggers Bax activation, resulting in selective apoptosis‐inducing factor (AIF) relocation in T lymphocytes entering the early commitment phase to apoptosis. J. Biol. Chem. 278:31401‐31411.
  Boya, P. and Kroemer 2008. Lysosomal membrane permeabilization in cell death. Oncogene 27:6434‐6451.
  Cirman, T., Oresić, K., Mazovec, G.D., Turk, V., Reed, J.C., Myers, R.M., Salvesen, G.S., and Turk, B. 2004. Selective disruption of lysosomes in HeLa cells triggers apoptosis mediated by cleavage of Bid by multiple papain‐like lysosomal cathepsins. J. Biol. Chem. 279:3578‐3587.
  Costantini, L.M., Gilberti, R.M., and Knecht, D.A. 2011. The phagocytosis and toxicity of amorphous silica. PloS One 6:e14647.
  de Duve, C. 1975. Exploring cells with a centrifuge. Science 189:186‐194.
  Düssmann, H., Rehm, M., Concannon, C.G., Anguissola, S., Würstle, M., Kacmar, S., Völler, P., Huber, H.J., and Prehn, J.H.M. 2010. Single‐cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation. Cell Death Differ. 17:278‐290.
  Gilberti, R.M., Joshi, G.N., and Knecht, D.A. 2008. The phagocytosis of crystalline silica particles by macrophages. Am. J. Respir. Cell Mol. Biol. 39:619‐627.
  Goldstein, J.C., Muñoz‐Pinedo, C., Ricci, J.‐E., Adams, S.R., Kelekar, A., Schuler, M., Tsien, R.Y., and Green, D.R. 2005. Cytochrome c is released in a single step during apoptosis. Cell Death Differ. 12:453‐462.
  He, L., Wu, X., Siegel, R., and Lipsky, P. 2006. TRAF6 regulates cell fate decisions by inducing caspase 8‐dependent apoptosis and the activation of NF‐kappaB. J. Biol. Chem. 281:11235‐11249.
  Joshi, G.N. and Knecht, D.A. 2013. Silica phagocytosis causes apoptosis and necrosis by different temporal and molecular pathways in alveolar macrophages. Apoptosis 18:271‐285.
  Kawai, H., Suzuki, T., Kobayashi, T., Mizuguchi, H., Hayakawa, T., and Kawanishi, T. 2004. Simultaneous imaging of initiator/effector caspase activity and mitochondrial membrane potential during cell death in living HeLa cells. Biochimica Biophysica Acta 1693:101‐110.
  Kerr, J.F., Wyllie, A.H., and Currie, A.R. 1972. Apoptosis: A basic biological phenomenon with wide‐ranging implications in tissue kinetics. Br. J. Cancer 26:239.
  Kim, H., Tu, H.‐C., Ren, D., Takeuchi, O., Jeffers, J.R., Zambetti, G.P., Hsieh, J.J.‐D., and Cheng, E.H.‐Y. 2009. Stepwise activation of BAX and BAK by tBID, BIM, and PUMA initiates mitochondrial apoptosis. Mol. Cell 36:487‐499.
  Lima, H., Jacobson, L.S., Goldberg, M.F., Chandran, K., Diaz‐Griffero, F., Lisanti, M.P., and Brojatsch, J. 2013. Role of lysosome rupture in controlling Nlrp3 signaling and necrotic cell death. Cell Cycle 12:1868‐1878.
  Loew, L.M., Tuft, R.A., Carrington, W., and Fay, F.S. 1993. Imaging in five dimensions: Time‐dependent membrane potentials in individual mitochondria. Biophys. J. 65:2396‐2407.
  Luo, X., Budihardjo, I., Zou, H., Slaughter, C., and Wang, X. 1998. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94:481‐490.
  McStay, G.P., Salvesen, G.S., and Green, D.R. 2008. Overlapping cleavage motif selectivity of caspases: Implications for analysis of apoptotic pathways. Cell Death Differ. 15:322‐331.
  Oltval, Z.N., Milliman, C.L., and Korsmeyer, S.J. 1993. Bcl‐2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programed cell death. Cell 74:609‐619.
  Persson, H.L. 2005. Iron‐dependent lysosomal destabilization initiates silica‐induced apoptosis in murine macrophages. Toxicol. Lett. 159:124‐133.
  Repnik, U. and Turk, B. 2010. Lysosomal‐mitochondrial cross‐talk during cell death. Mitochondrion 10:662‐669.
  Schneider, C.A., Rasband, W.S., and Eliceiri, K.W. 2012. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9:671‐675.
  Slee, E.A., Harte, M.T., Kluck, R.M., Wolf, B.B., Casiano, C.A., Newmeyer, D.D., Wang, H.G., Reed, J.C., Nicholson, D.W., Alnemri, E.S., Green, D.R., and Martin, S.J. 1999. Ordering the cytochrome c‐initiated caspase cascade: Hierarchical activation of caspases‐2, ‐3, ‐6, ‐7, ‐8, and ‐10 in a caspase‐9‐dependent manner. J. Cell Biol. 144:281‐292.
  Tait, S.W.G. and Green, D.R. 2010. Mitochondria and cell death: Outer membrane permeabilization and beyond. Nat. Rev. Mol. Cell Biol. 11:621‐632.
  Thibodeau, M.S., Giardina, C., Knecht, D.A., Helble, J., and Hubbard, A.K. 2004. Silica‐induced apoptosis in mouse alveolar macrophages is initiated by lysosomal enzyme activity. Toxicol. Sci. 80:34‐48.
  Thiele, D.L. and Lipsky, P.E. 1990. Mechanism of L‐leucyl‐L‐leucine methyl ester‐mediated killing of cytotoxic lymphocytes: Dependence on a lysosomal thiol protease, dipeptidyl peptidase I, that is enriched in these cells. Proc. Natl. Acad. Sci. U.S.A. 87:83‐87.
  Tyas, L., Brophy, V.A., Pope, A., Rivett, A.J., and Tavaré, J.M. 2000. Rapid caspase‐3 activation during apoptosis revealed using fluorescence‐resonance energy transfer. EMBO Rep. 1:266‐270.
  Vaux, D.L., Cory, S., and Adams, J.M. 1988. Bcl‐2 gene promotes haemopoietic cell survival and cooperates with c‐myc to immortalize pre‐B cells. Nature 335:440‐442.
  Wang, K., Yin, X.‐M., Chao, D.T., Milliman, C.L., and Korsmeyer, S.J. 1996. BID: A novel BH3 domain‐only death agonist. Genes Dev. 10:2859‐2869.
  Yang, E., Zha, J., Jockel, J., Boise, L.H., Thompson, C.B., and Korsmeyer, S.J. 1995. Bad, a heterodimeric partner for Bcl‐XL and Bcl‐2, displaces Bax and promotes cell death. Cell 80:285‐291.
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