A Violet Ratiometric Membrane Probe for the Detection of Apoptosis

William G. Telford1

1 Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.38
DOI:  10.1002/0471142956.cy0938s59
Online Posting Date:  January, 2012
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Abstract

This unit describes the use of a novel violet‐excited membrane‐binding probe F2N12S [4′‐N,N‐diethylamino‐6‐(N,N,N‐dodecyl‐methylamino‐sulfopropyl)‐methyl‐3‐hydroxyflavone] for the flow cytometric detection of the changes in membrane asymmetry, fluidity, and charge that accompanies apoptosis. This reagent inserts into the plasma membrane and undergoes a shift in emission from orange to green during the membrane alterations that occur during apoptosis. Since its mechanism of action differs from annexin V, it can be used in situations where annexin V binding is problematic, as in adherent cells removed from their growth substrate. Like annexin V, it can also be readily combined with other flow cytometric assays for apoptosis, allowing detailed multiparametric analysis of the apoptotic process. Curr. Protoc. Cytom. 59:9.38.1‐9.38.12. © 2012 by John Wiley & Sons, Inc.

Keywords: apoptosis; membrane probe; annexin V; F2N12S

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Labeling with F2N12S and DNA Dye Alone
  • Alternate Protocol 1: Labeling with F2N12S and Multiple Apoptosis Reagents
  • Alternate Protocol 2: Labeling with F2N12S, Annexin V, and DNA Dyes
  • Alternate Protocol 3: Labeling with F2N12S, Caspase Substrates, Annexin V, and DNA Dyes
  • Basic Protocol 2: Flow Cytometer Setup, Data Acquisition, and Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Labeling with F2N12S and DNA Dye Alone

  Materials
  • F2N12S reagent (Invitrogen Life Technologies, cat. no. A35137): sold as a solution and is stored in anhydrous conditions at minus 20°C; it can withstand multiple freeze/thaw cycles without harm
  • DNA dyes including:
    • Propidium iodide (PI; see recipe)
    • 7‐aminoactinomycin D (7‐AAD; see recipe)
  • Cells (this reagent has been tested for a variety of cell lines and primary cell types; EL4 mouse lymphoma cells have been used in the examples shown)
  • Standard Hank's balanced salt solution (HBSS) containing calcium and magnesium and no phenol red makes a good incubation buffer for F2N12S (this buffer should not be confused with wash buffer below, which can be used for steps preceding F2N12S labeling)
  • Fluorescent alignment verification microsphere standard, to verify violet laser alignment: Single intensity Rainbow Ultra beads (Spherotech) or AlignFlow UV beads (Invitrogen) are well‐excited with violet lasers and are good for alignment verification; rainbow 8‐peak (Spherotech) or InSpeck Blue (Invitrogen) multi‐intensity bead cocktails are also good for monitoring laser alignment, since degradation in low‐signal resolution can be observed
  • Flow cytometer equipped with a violet laser diode or other violet laser (e.g., F2N12S can be readily carried out on BD Bioscience LSR II, LSR Fortessa, FACSVerse, Influx, and FACSAria series cytometers, Beckman‐Coulter Gallios and Astrios, Stratedigm S series, Miltenyi Biotec MACSQuant, Partec CyFlow series, the Life Technologies/Applied Biosystems Attune, and Cytek Development modified BD instruments with a violet laser)

Alternate Protocol 1: Labeling with F2N12S and Multiple Apoptosis Reagents

  • Wash buffer
  • Annexin V (F2N12S can be readily combined with almost any annexin V conjugates that are not UV or violet excited, with probes such as Alexa Fluor 405 and Pacific Blue annexin V being excluded; FITC, PE, PE‐Cy5, APC, and Cy5 or Alexa Fluor 647 are all commercially available and are compatible with F2N12S)
  • Centrifuge
NOTE: A wash buffer composed of colorless RPMI‐1640 with 2% fetal bovine serum makes a good generic wash and incubation buffer for labeling steps preceding F2N12S incubation. This buffer contains calcium and magnesium and is compatible with annexin V labeling. It should be washed out with the same standard HBSS containing no protein used for F2N12S prior to F2N12S labeling.

Alternate Protocol 2: Labeling with F2N12S, Annexin V, and DNA Dyes

  • Fluorogenic caspase substrates including:
  • PhiPhiLux (Oncoimmunin)
  • NucView 488 (Biotium)
  • CellEvent (Invitrogen Life Technologies)

Alternate Protocol 3: Labeling with F2N12S, Caspase Substrates, Annexin V, and DNA Dyes

  Materials
  • Positive and negative controls
  • Fluorogenic caspase substrates including:
    • PhiPhiLux (Oncoimmunin)
    • NucView 488 (Biotium)
    • CellEvent (Invitrogen Life Technologies)
  • Annexin V (F2N12S can be readily combined with almost any annexin V conjugates that are not UV or violet excited, with probes such as Alexa Fluor 405 and Pacific Blue annexin V being excluded; FITC, PE, PE‐Cy5, APC, and Cy5 or Alexa Fluor 647 are all commercially available and are compatible with F2N12S)
  • Flow cytometer equipped with a violet laser diode or other violet laser (e.g., F2N12S can be readily carried out on BD Bioscience LSR II, LSR Fortessa, FACSVerse, Influx, and FACSAria series cytometers, Beckman‐Coulter Gallios and Astrios, Stratedigm S series, Miltenyi Biotec MACSQuant, Partec CyFlow series, the Life Technologies/Applied Biosystems
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Figures

Videos

Literature Cited

Literature Cited
   Bedner, E. Smolewski, P., Amstad, P., and Darzynkiewicz, Z. 2000. Activation of caspases measured in situ by binding of fluorochrome‐labeled inhibitors of caspases (FLICA); correlation with DNA fragmentation. Exp. Cell Res. 260:308‐313.
   Das, R., Klymchenko, A.S., Duportail, G., and Mély, Y. 2008. Excited state proton transfer and solvent relaxation of a 3‐hydroxyflavone probe in lipid bilayers. J. Phys. Chem. B 112:11929‐11935.
   Hope‐Roberts, M., Horobin, R.W., and Wainwright, M. 2011. Identifying apoptotic cells with the 3‐hydroxyflavone derivative F2N12S, a ratiometric fluorescent small molecule probe selective for plasma membranes: A possible general mechanism for selective uptake into apoptotic cells. Biotech. Histochem. 86:255‐261.
   Klymchenko, A.S., Oncul, S., Didier, P., Schaub, E., Bagatolli, L., Duportail, G., and Mély, Y. 2009. Visualization of lipid domains in giant unilamellar vesicles using an environment‐sensitive membrane probe based on 3‐hydroxyflavone. Biochim. Biophys. Acta 1788:495‐499.
   Komoriya, A., Packard, B.Z., Brown, M.J., Wu, M.L., and Henkart, P.A. 2000. Assessment of caspase activities in intact apoptotic thymocytes using cell‐permeable fluorogenic caspase substrates. J. Exp. Med. 191:1819‐1828.
   Kułakowska, A., Jurkiewicz, P., Sýkora, J., Benda, A., Mely, Y., and Hof, M. 2010. Fluorescence lifetime tuning—A novel approach to study flip‐flop kinetics in supported phospholipid bilayers. J. Fluoresc. 20:563‐569.
   Oncul, S., Klymchenko, A.S., Kucherak, O.A., Demchenko, A.P., Martin, S., Dontenwill, M., Arntz, Y., Didier, P., Duportail, G., and Mély, Y. 2010. Liquid ordered phase in cell membranes evidenced by a hydration‐sensitive probe: Effects of cholesterol depletion and apoptosis. Biochim. Biophys. Acta 1798:1436‐1443.
   Roche, Y., Klymchenko, A.S., Gerbeau‐Pissot, P., Gervais, P., Mély, Y., Simon‐Plas, F., and Perrier‐Cornet, J.M. 2010. Behavior of plant plasma membranes under hydrostatic pressure as monitored by fluorescent environment‐sensitive probes. Biochim. Biophys. Acta 1798:1601‐1607.
   Shynkar, V.V., Klymchenko, A.S., Kunzelmann, C., Duportail, G., Muller, C.D., Demchenko, A.P., Freyssinet, J.M., and Mely, Y. 2007. Fluorescent biomembrane probe for ratiometric detection of apoptosis. J. Am. Chem. Soc. 129:2187‐2193.
   Telford, W.G., Komoriya, A., Packard, B.Z., and Bagwell, C.B. 2010. Analysis of apoptosis by flow and image cytometry. In Methods in Molecular Biology, Volume 699, Flow Cytometry Protocols, 4th Ed. (T.S. Hawley and R.G. Hawley, eds.) pp. 203‐228. Humana Press, London.
   van Engeland, M., Nieland, L.J., Ramaekers, F.C., Schutte, B., and Reutelingsperger, C.P. 1998. Annexin V‐affinity assay: A review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry 31:1‐9.
   Vermes, I., Haanen, C., Steffens‐Nakken, H., and Reutelingsperger, C. 1995. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin V. J. Immunol. Methods 184:39‐51.
   Wlodkowic, D., Telford, W.G., Skommer, J., and Darzynkiewicz, Z. 2011. Apoptosis and beyond: Cytometry in studies of programmed cell death. In Methods in Cell Biology, Volume 103 (Z. Darzynkiewicz, E. Holden, A. Orfao, W.G. Telford, and D. Wlodkowic, eds.) pp. 55‐98. Academic Press, New York.
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