SYTO Probes: Markers of Apoptotic Cell Demise

Donald Wlodkowic1, Joanna Skommer2

1 University of Glasgow, UK, 2 University of Kuopio, Finland
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.33
DOI:  10.1002/0471142956.cy0733s42
Online Posting Date:  October, 2007
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

As mechanistic studies on tumor cell death advance towards their ultimate translational goal, there is a need for specific, rapid, and high‐throughput analytical tools to detect diverse cell demise modes. Patented DNA‐binding SYTO probes, for example, are gaining increasing interest as easy‐to‐use markers of caspase‐dependent apoptotic cell death. They are proving convenient for tracking apoptosis in diverse hematopoietic cell lines and primary tumor samples, and, due to their spectral characteristics, appear to be useful for the development of multiparameter flow cytometry assays. Herein, several protocols for multiparametric assessment of apoptotic events using SYTO probes are provided. There are protocols describing the use of green fluorescent SYTO 16 and red fluorescent SYTO 17 dyes in combination with plasma membrane permeability markers. Another protocol highlights the multiparametric use of SYTO 16 dye in conjunction with the mitochondrial membrane potential sensitive probe, tetramethylrhodamine methyl ester (TMRM), and the plasma membrane permeability marker, 7‐aminoactinomycin D (7‐AAD). Curr. Protocol. Cytom. 42:7.33.1‐7.33.12. © 2007 by John Wiley & Sons, Inc.

Keywords: SYTO; apoptosis; flow cytometry; multi‐parameter assay

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Assessment of Apoptosis Using SYTO Probes and Plasma Membrane Permeability Markers
  • Basic Protocol 1: SYTO 16 and Propidium Iodide Staining
  • Alternate Protocol 1: SYTO 16 and 7‐Amino Actinomycin D (7‐AAD) Staining
  • Alternate Protocol 2: SYTO 17 and Propidium Iodide Staining
  • Alternate Protocol 3: SYTO 17 and YO‐PRO 1 Staining
  • Alternate Protocol 4: Multiparametric Assessment of Apoptosis Using SYTO 16, Mitochondrial Membrane Potential (Δψm) Sensitive Probe TMRM, and Plasma Membrane Permeability Marker 7‐AAD
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: SYTO 16 and Propidium Iodide Staining

  Materials
  • Cell suspension in appropriate culture medium
  • PBS ( appendix 2A)
  • 10 µM SYTO 16 working solution in PBS (see recipe)
  • 50 µg/ml propidium iodide stock solution in PBS (PI; see recipe)
  • 30 mM Verapamil (P‐gp inhibitor; see recipe), optional
  • FACS buffer (see recipe), ice cold
  • 12 × 75–mm polystyrene FACS tubes
  • Tabletop centrifuge
  • Flow cytometer

Alternate Protocol 1: SYTO 16 and 7‐Amino Actinomycin D (7‐AAD) Staining

  • 1 mg/ml 7‐AAD stock solution (see unit 9.2)

Alternate Protocol 2: SYTO 17 and Propidium Iodide Staining

  • 5 µM SYTO 17 working solution in PBS (see recipe)

Alternate Protocol 3: SYTO 17 and YO‐PRO 1 Staining

  • 10 µM YO‐PRO 1 working solution in PBS (see recipe)

Alternate Protocol 4: Multiparametric Assessment of Apoptosis Using SYTO 16, Mitochondrial Membrane Potential (Δψm) Sensitive Probe TMRM, and Plasma Membrane Permeability Marker 7‐AAD

  • 1 µM TMRM working solution in PBS (see recipe)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Broxterman, H.J., Schuurhuis, G.J., Lankelma, J., Oberink, J.W., Eekman, C.A., Claessen, A.M., Hoekman, K., Poot, M., and Pinedo, H.M. 1997. Highly sensitive and specific detection of P‐glycoprotein function for haematological and solid tumour cells using a novel nucleic acid stain. Br. J. Cancer 76:1029‐1034.
   Darzynkiewicz, Z., Juan, G., Li, X., Gorczyca, W., Murakami, T., and Traganos, F. 1997. Cytometry in cell necrobiology: Analysis of apoptosis and accidental cell death (necrosis). Cytometry 27:1‐20.
   Darzynkiewicz, Z., Li, X., and Bedner, E. 2001. Use of flow and laser‐scanning cytometry in analysis of cell death. Methods Cell Biol. 66:69‐109.
   Eray, M., Matto, M., Kaartinen, M., Andersson, L., and Pelkonen, J. 2001. Flow cytometric analysis of apoptotic subpopulations with a combination of annexin V‐FITC, propidium iodide, and SYTO 17. Cytometry 43:134‐142.
   Frey, T. 1995. Nucleic acid dyes for detection of apoptosis in live cells. Cytometry 21:265‐274.
   Huang, X., Halicka, H.D., Traganos, F., Tanaka, T., Kurose, A., and Darzynkiewicz, Z. 2005. Cytometric assessment of DNA damage in relation to cell cycle phase and apoptosis. Cell Prolif. 38:223‐243.
   Idziorek, T., Estaquier, J., De Bels, F., and Ameisen, J.C. 1995. YOPRO‐1 permits cytofluorometric analysis of programmed cell death (apoptosis) without interfering with cell viability. J. Immunol. Methods 185:249‐258.
   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 26:239‐257.
   Leist, M. and Jäättela, M. 2001. Four deaths and a funeral: From caspases to alternative mechanisms. Nat. Rev. Mol. Cell Biol. 2:589‐598.
   Poot, M., Gibson, L.L., and Singer, V.L. 1997. Detection of apoptosis in live cells by MitoTracker red CMXRos and SYTO dye flow cytometry. Cytometry 27:358‐364.
   Pozarowski, P., Huang, X., Halicka, D.H., Lee, B., Johnson, G., and Darzynkiewicz, Z. 2003. Interactions of fluorochrome‐labeled caspase inhibitors with apoptotic cells: A caution in data interpretation. Cytometry 55:50‐60.
   Schuurhuis, G.J., Muijen, M.M., Oberink, J.W., de Boer, F., Ossenkoppele, G.J., and Broxterman, H.J. 2001. Large populations of non‐clonogenic early apoptotic CD34‐positive cells are present in frozen‐thawed peripheral blood stem cell transplants. Bone Marrow Transplant. 27:487‐498.
   Serafeim, A., Grafton, G., Chamba, A., Gregory, C.D., Blakely, R.D., Bowery, N.G., Barnes, N.M., and Gordon, J. 2002. 5‐Hydroxytryptamine drives apoptosis in biopsylike Burkitt lymphoma cells: Reversal by selective serotonin reuptake inhibitors. Blood 99:2545‐2553.
   Serafeim, A., Holder, M.J., Grafton, G., Chamba, A., Drayson, M.T., Luong, Q.T., Bunce, C.M., Gregory, C.D., Barnes, N.M., and Gordon, J. 2003. Selective serotonin reuptake inhibitors directly signal for apoptosis in biopsy‐like Burkitt lymphoma cells. Blood 101:3212‐3219.
   Skommer, J., Wlodkowic, D., Matto, M., Eray, M., and Pelkonen, J. 2006. HA14‐1, a small molecule Bcl‐2 antagonist, induces apoptosis and modulates action of selected anticancer drugs in follicular lymphoma B cells. Leuk. Res. 30:322‐331.
   Sparrow, R.L. and Tippett, E. 2005. Discrimination of live and early apoptotic mononuclear cells by the fluorescent SYTO 16 vital dye. J. Immunol. Methods. 305:173‐187.
   van der Pol, M.A., Broxterman, H.J., Westra, G., Ossenkoppele, G.J., and Schuurhuis, G.J. 2003. Novel multiparameter flow cytometry assay using Syto16 for the simultaneous detection of early apoptosis and apoptosis‐corrected P‐glycoprotein function in clinical samples. Cytometry B Clin. Cytom. 55:14‐21.
   van Zandvoort, M.A., de Grauw, C.J., Gerritsen, H.C., Broers, J.L., oude Egbrink, M.G., Ramaekers, F.C., and Slaaf, D.W. 2002. Discrimination of DNA and RNA in cells by a vital fluorescent probe: Lifetime imaging of SYTO13 in healthy and apoptotic cells. Cytometry A. 47:226‐235.
   Westers, T.M., Houtenbos, I., Schuurhuis, G.J., Ossenkoppele, G.J., and van de Loosdrecht, A. 2005. Quantification of T‐cell‐mediated apoptosis in heterogeneous populations using four‐color multiparameter flow cytomety. Cytometry A 66:71‐77.
   Wlodkowic, D., Skommer, J., and Pelkonen, J. 2007. Towards an understanding of apoptosis detection by SYTO dyes. Cytometry A 71A:61‐72.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library