CyTOF Mass Cytometry for Click Proliferation Assays

Vinko Tosevski1, Egor Ulashchik2, Andrea Trovato3, Paolo Cappella3

1 Mass Cytometry Facility, University of Zurich, Zurich, 2 Primetech ALC, Minsk, 3 FlowMetric Europe SpA, Lodi, Milan
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.50
DOI:  10.1002/cpcy.25
Online Posting Date:  July, 2017
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Novel cell analyzers, including polychromatic flow cytometers and isotopical cytometry by time of flight (CyTOF) mass cytometers, enable simultaneous measurement of virtually bondless characteristics at the single‐cell level. BrdU assays for quantifying cellular proliferation are common but have several limitations, including the need for a DNA denaturation step and inability to simultaneously resolve multiple parameters and phenotypic complexity. Click chemistry reactions have become popular in the past decade, as they can resolve these issues. This protocol introduces a novel assay able to bridge flow cytometry and CyTOF analysis for active S‐phase determination in cell cycle applications, combining well‐established click chemistry with a novel iodo‐deoxyuridine (IdU) azide derivative and a cross‐reactive anti‐IdU antibody for detecting incorporated EdU during DNA synthesis. This method is preferred over traditional BrdU‐based assays for complex and multiparametric experiments. It provides a feasible cost‐effective approach for detecting ethynyl‐labeled nucleotides, with the advantage of combining flow and mass cytometry analyses. © 2017 by John Wiley & Sons, Inc.

Keywords: BrdU; click chemistry; cell cycle; CyTOF; IdU azide; IMA; mass cytometry

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Table of Contents

  • Introduction
  • Basic Protocol 1: Click Chemistry Using 5′‐Azido‐IdU for Ethynyl Uridine Detection by Anti‐IdU Antibody and 127I Detection
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Click Chemistry Using 5′‐Azido‐IdU for Ethynyl Uridine Detection by Anti‐IdU Antibody and 127I Detection

  • Suspension cell culture (e.g., Jurkat cells) in appropriate culture medium
  • Ethynyl nucleotide (select one):
    • 10 mM EdU (Berry & Associates, PY7562) in DMSO
    • 10 mM EdC (Sigma‐Aldrich, T511307) in DMSO
    • 40 mM F‐ara‐EdU (Sigma‐Aldrich, T511293) in DMSO
  • Control nucleotides:
    • 3 mM BrdU (Sigma‐Aldrich, B5002) in water
    • 25 mM IdU (Berry & Associates, PY7615) in DMSO
    • 25 mM IdC (Berry & Associates, PY7607) in DMSO
  • Phosphate‐buffered saline (PBS) without calcium and magnesium (Thermo Fisher Scientific)
  • 100% methanol (Sigma‐Aldrich)
  • BSA Stain Buffer (BD Biosciences)
  • 0.1% Triton X‐100 (Sigma) in PBS
  • 10 mM 5‐iodo‐5′‐azido‐2′,5′‐dideoxyuridine (IMA; HPLC >97%) in DMSO
  • 10 mM 5‐bromo‐3′‐azido‐2′,3′‐dideoxyuridine (BMA; Berry & Associates, PY7286) in DMSO
  • 0.1 M copper(II) sulfate solution (Fluka, 35185)
  • 0.5 M sodium‐L‐ascorbate (see recipe; also see unit 7.34), freshly prepared
  • SBT: BSA Stain Buffer with 0.5% Tween‐20 (Sigma‐Aldrich), freshly prepared
  • Antibodies:
    • 0.025 mg/ml anti‐BrdU monoclonal mouse antibody (clone B44, BD Biosciences)
    • Alexa Fluor 488–conjugated goat anti‐mouse IgG (H+L, Thermo Fisher Scientific, A‐11001)
  • PI staining solution (see recipe)
  • Cell‐ID Intercalator‐Ir (Fluidigm, 201192 A or 201192B)
  • Cell‐ID Cisplatin (Fluidigm, 201064)
  • 15‐ml screw‐cap centrifuge tubes
  • Vacuum aspirator (e.g., Costar)
  • Hemocytometer
  • Shaker
  • 5‐ml (12 × 75–mm) polystyrene tubes (BD Falcon) for FCM
  • Flow cytometer with excitation at 488 nm (e.g., Beckton Dickinson BD FACSAria III)
  • 2‐ml microcentrifuge tubes
  • MaxPar Cell Staining Buffer (Fluidigm, 201068)
  • MaxPar Fix and Perm Buffer (Fluidigm, 201067)
  • Cell‐ID Cisplatin (Fluidigm, 201064, optional)
  • Cell‐ID Intercalator‐Ir (Fluidigm, 201192 A or 201192B)
  • EQ Four Element Calibration Beads (Fluidigm, 201078)
  • 5‐ml (12 × 75–mm) polystyrene round‐bottom tubes with cell‐strainer caps (BD Falcon, 352235)
  • CyTOF mass cytometer (e.g., CyTOF 2, Fluidigm)
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Internet Resources
  Fluidigm website.
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