Multiparameter Analysis of Apoptosis Using Lab‐on‐a‐Chip Flow Cytometry

Donald Wlodkowic1, Joanna Skommer2, Jin Akagi2, Yoo Fujimura3, Kazuo Takeda3

1 RMIT University, Melbourne, 2 University of Auckland, Auckland, 3 R&D Division, On‐chip Biotechnologies, Tokyo
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.42
DOI:  10.1002/0471142956.cy0942s66
Online Posting Date:  October, 2013
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Abstract

The age of microfluidic flow cytometry (µFCM) is fast becoming a reality. One of the most exciting applications of miniaturized chip‐based cytometers is multivariate analysis using sampling volumes as small as 10 µl while matching the multiparameter data collection of conventional flow cytometers. We outline several innovative protocols for analyzing caspase‐dependent cell death and cell cycle (DNA‐content) profile using a fully integrated microfluidic flow cytometry system, Fishman‐R. The first protocol describes the use of a new plasma membrane–permeability marker, DRAQ7, and the fluorogenic caspase substrate PhiPhiLux to track caspase activation during programmed cell death. Also outlined is the use of DRAQ7 fluorochrome in conjunction with the mitochondrial membrane potential–sensitive probe TMRM to track dissipation of inner mitochondrial cross‐membrane potential. Another protocol adds the ability to measure dissipation of mitochondrial inner membrane potential (using TMRM probe) in relation to the cell cycle profile (using DRAQ5 probe) in living leukemic cells. Finally, we describe the combined use of fluorogenic caspases substrate PhiPhiLux with DRAQ5 probe to measure caspase activation in relation to the cell cycle profile in living tumor cells. Curr. Protoc. Cytom. 66:9.42.1‐9.42.15. © 2013 by John Wiley & Sons, Inc.

Keywords: microfluidics; Lab‐on‐a‐Chip; flow cytometry; cell cycle; apoptosis; multiparameter; assay

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Two‐Color Analysis of Caspase‐Dependent Apoptosis Using DRAQ7 AND PhiPhiLux Probes
  • Alternate Protocol 1: Dissipation of the Mitochondrial Inner Membrane Potential ΔΨm Sensitive Probe TMRM and Plasma Integrity Marker DRAQ7
  • Alternate Protocol 2: Analysis of Caspase Activation in Relation to Cell Cycle Phase Using PhiPhiLux AND DRAQ5 Probes
  • Alternate Protocol 3: Analysis of Dissipation of Mitochondrial Inner Membrane Potential (ΔΨm) in Relation to Cell Cycle Phase Using TMRM and DRAQ5 Probes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Two‐Color Analysis of Caspase‐Dependent Apoptosis Using DRAQ7 AND PhiPhiLux Probes

  Materials
  • Cell suspension in appropriate culture medium
  • 10 µM PhiPhiLux G 1D 2 (OncoImmunin Inc., http://phiphilux.com/) stock solution in RPMI 1640 medium (store vials protected from light at 4°C; after opening store vials at −10° to −20°C)
  • 30 µM DRAQ7 (BioStatus Ltd., cat. no. DR7100, http://www.biostatus.com/) stock solution in RPMI 1640 medium (store protected from light at 4°C)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Appropriate culture medium for cells
  • Fishman‐R microfluidic chip (On‐chip Biotechnologies, http://www.on‐chip.co.jp)
  • Fishman‐R microfluidic analyzer (On‐chip Biotechnologies, http://www.on‐chip.co.jp)

Alternate Protocol 1: Dissipation of the Mitochondrial Inner Membrane Potential ΔΨm Sensitive Probe TMRM and Plasma Integrity Marker DRAQ7

  Additional Materials (also see protocol 1Basic Protocol)
  • 10 µM TMRM working solution (see recipe)

Alternate Protocol 2: Analysis of Caspase Activation in Relation to Cell Cycle Phase Using PhiPhiLux AND DRAQ5 Probes

  Additional Materials (also see protocol 1Basic Protocol)
  • 5 mM DRAQ5 (Biostatus Ltd., cat. no. DR50050, http://www.biostatus.com/) stock solution in water (store protected from light at 4°C)

Alternate Protocol 3: Analysis of Dissipation of Mitochondrial Inner Membrane Potential (ΔΨm) in Relation to Cell Cycle Phase Using TMRM and DRAQ5 Probes

  Additional Materials (also see protocol 2)
  • 10 µM TMRM working solution (see recipe)
  • 5 mM DRAQ5 (Biostatus Ltd., cat. no. DR50050, http://www.biostatus.com/) stock solution in water (store protected from light at 4°C)
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Figures

Videos

Literature Cited

Literature Cited
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