Kinetic Viability Assays Using DRAQ7 Probe

Donald Wlodkowic1, Jin Akagi2, Jurek Dobrucki3, Rachel Errington4, Paul J Smith4, Kazuo Takeda5, Zbigniew Darzynkiewicz6

1 The BioMEMS Research Group, School of Applied Sciences, RMIT University, Melbourne, Australia, 2 The BioMEMS Research Group, School of Chemical Sciences, University of Auckland, Auckland, New Zealand, 3 Division of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland, 4 School of Medicine, Cardiff University, Cardiff, United Kingdom, 5 R&D Division, On‐Chip Biotechnologies, Tokyo, Japan, 6 Brander Cancer Research Institute and Department of Pathology, New York Medical College, Valhalla, New York
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.41
DOI:  10.1002/0471142956.cy0941s65
Online Posting Date:  July, 2013
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Abstract

Cell death within cell populations is a stochastic process where cell‐to‐cell variation in temporal progression through the various stages of cell death arises from asynchrony of subtle fluctuations in the signaling pathways. Most cell death assays rely on detection of the specific marker of cell demise at the end‐point of cell culturing. Such an approach cannot account for the asynchrony and the stochastic nature of cell response to the death‐inducing signal. There is a need therefore for rapid and high‐throughput bioassays capable of continuously tracking viability of individual cells from the time of encountering a stress signal up to final stages of their demise. In this context, a new anthracycline derivative, DRAQ7, is gaining increasing interest as an easy‐to‐use marker capable of long‐term monitoring of cell death in real‐time. This novel probe neither penetrates the plasma membrane of living cells nor does it affect the cells' susceptibility to the death‐inducing agents. However, when the membrane integrity is compromised, DRAQ7 enters cells undergoing demise and binds readily to nuclear DNA to report cell death. Here, we provide three sets of protocols for viability assays using DRAQ7 probe. The first protocol describes the innovative use of single‐color DRAQ7 real‐time assay to dynamically track cell viability. The second protocol outlines a simplified end‐point DRAQ7 staining approach. The final protocol highlights the real‐time and multiparametric apoptosis assay utilizing DRAQ7 dye concurrently with tetramethylrhodamine methyl ester (TMRM), the mitochondrial trans‐membrane electrochemical potential (ΔΨm) sensing probe. Curr. Protoc. Cytom. 65:9.41.1‐9.41.8. © 2013 by John Wiley & Sons, Inc.

Keywords: viability; cell death; real‐time assay; DRAQ7; flow cytometry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Kinetic Analysis of Cell Viability Using DRAQ7 Probe
  • Alternate Protocol 1: Analysis of Cell Viability Using End‐Point DRAQ7 Staining
  • Alternate Protocol 2: Real‐Time Multiparametric Assessment of Cell Death Using DRAQ7 and Mitochondrial Membrane Potential (ΔΨm) Sensitive Probe TMRM
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Kinetic Analysis of Cell Viability Using DRAQ7 Probe

  Materials
  • Cell suspension in appropriate culture medium
  • 30 µM DRAQ7 stock solution (store protected from light at 4°C)
  • Cell culture vessels (e.g., microtiter plate or cell culture flask)
  • 37°C humidified incubator
  • 12 × 75–mm polystyrene FACS tubes or 1.5 ml microcentrifuge tubes (as appropriate)
  • Flow cytometer
CAUTION: DRAQ7 probe is a DNA‐binding molecule and thus can be considered as a potential carcinogen. Always use gloves when handling DRAQ7 solutions.

Alternate Protocol 1: Analysis of Cell Viability Using End‐Point DRAQ7 Staining

  • 10 µM TMRM working solution in appropriate cell culture medium (see recipe)
CAUTION: Although there are no reports on TMRM toxicity, appropriate precautions should always be applied when handling TMRM solutions.
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Figures

  •   FigureFigure 9.41.1 Assessment of live, early apoptotic and late apoptotic/necrotic cells based on stainability with plasma membrane permeability marker DRAQ7. Analysis was based on real‐time labeling of THP‐1 cells with 3 µM DRAQ7. Bivariate dot plots DRAQ7 versus forward scatter (FS) enable discrimination of live (DRAQ7; green gate), early apoptotic (DRAQ7dim; blue gate), and late apoptotic/necrotic (DRAQ7+; red gate) subpopulations. DRAQ7 probe was excited using a 633‐nm laser and logarithmically amplified fluorescence signals were collected using a 660‐nm long‐pass filter.
  •   FigureFigure 9.41.2 Discrimination of viable, apoptotic and late apoptotic/necrotic cells based on the ΔΨm marker tetramethylrhodamine methyl ester (TMRM) and plasma membrane permeability marker DRAQ7. Analysis was based on real‐time labeling of THP‐1 cells with 3 µM DRAQ7 and 150 nm TMRM. Bivariate dot plots DRAQ7 versus TMRM enable discrimination of live (TMRM+/DRAQ7; green gate), early apoptotic (TMRMlow/DRAQ7dim; blue gate), and late apoptotic/necrotic (TMRMlow/DRAQ7+; red gate) subpopulations. DRAQ7 probe was excited using a 633‐nm laser and logarithmically amplified fluorescence signals were collected using a 660‐nm long‐pass filter. TMRM was excited using a 488‐nm laser and logarithmically amplified fluorescence signals were collected using a 575‐nm long‐pass filter. Debris signals were excluded electronically by setting the proper low threshold.

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Literature Cited

Literature Cited
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   Darzynkiewicz, Z., Staiano‐Coico, L., and Melamed, M.R. 1981. Increased mitochondrial uptake of rhodamine 123 during lymphocyte stimulation. Proc. Natl. Acad. Sci. U.S.A. 78:2383‐2387.
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   Zhao, H., Traganos, F., Dobrucki, J., Wlodkowic, D., and Darzynkiewicz, Z. 2009. Induction of DNA damage response by the supravital probes of nucleic acids. Cytometry A 75:510‐519.
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