High‐Dimensional Single‐Cell Analysis with Mass Cytometry

Tess Melinda Brodie1, Vinko Tosevski1

1 University of Zurich, Mass Cytometry Facility, Zurich
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 5.11
DOI:  10.1002/cpim.31
Online Posting Date:  August, 2017
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Abstract

Mass cytometry is an analytical technology that combines the sample preparation workflow typical of flow cytometry and the detection capacity of atomic mass spectroscopy, allowing for highly multiplexed measurements of protein or nucleic acid targets on single cells. In 2014, the mass cytometer was adapted for the acquisition of samples from microscopy slides (termed imaging mass cytometry), greatly increasing the applicability of this technology. By using antibodies (or other probes) labeled with purified metal isotopes, the mass cytometer is able to detect up to 50 different parameters (current practical limit) at the single‐cell level, enabling a deep and thorough profiling of individual cells in terms of their cell surface protein phenotype, physiological state, proliferation potential, and many other cell states or features. © 2017 by John Wiley & Sons, Inc.

Keywords: mass cytometry; CyTOF; Helios; high‐dimensional single‐cell analysis; T cells; immunophenotyping

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: 30‐Parameter Human Immunphenotyping Panel
  • Support Protocol 1: Metal Content Determination for In‐House Antibody Conjugates
  • Basic Protocol 2: 33‐Parameter Human Immunophenotyping Panel with Cytoplasmic Targets
  • Alternate Protocol 1: 33‐Parameter Human Immunophenotyping Panel with Cytoplasmic and Nuclear Targets
  • Basic Protocol 3: Titration of Mass Cytometry Barcoding Reagents
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: 30‐Parameter Human Immunphenotyping Panel

  Materials
  • 10 ml of human whole blood
  • Ficoll‐Pacque PLUS (GE Healthcare Life Sciences, cat. no. 17‐1440‐03)
  • Freezing medium: heat‐inactivated fetal bovine serum (FBS; Gibco, cat. no. 16140071) containing 10% dimethylsulfoxide (DMSO; Sigma‐Aldrich, cat. no. D8418)
  • Liquid N 2
  • Wash medium: RPMI 1640 medium (Gibco, cat. no. 11875085) containing 10% fetal bovine serum (FBS; Gibco, cat. no. 16140071)
  • Phosphate‐buffered saline (PBS; Gibco, cat. no. 10010023)
  • Cell‐ID cisplatin (Fluidigm, cat. no. 201064; dilute 1:5000 in PBS to prepare working solution)
  • Maxpar cell staining buffer (Fluidigm, cat. no. 201068)
  • Maxpar metal‐conjugated and validated antibodies (Table 5.11.1)
  • Cell‐ID intercalator‐Ir [Fluidigm, cat. no. 201192A (125 μM) or 201192B (500 μM)]: dilute to 100 nM iridium in Maxpar fix and perm buffer (Fluidigm, cat. no. 201067)
  • Maxpar water (Fluidigm, cat. no. 201069)
  • EQ Four Element Calibration beads (Fluidigm, cat. no. 201078)
  • SepMateTM‐ 50‐ml tubes (STEMCELL Technologies, cat. no. 15450)
  • 1.5‐ml Eppendorf‐style microcentrifuge tubes (Sigma‐Aldrich, cat. no. T9661)
  • 5‐ml round‐bottom polystyrene test tubes with cell strainer snap caps (Corning, cat. no. 352235)
  • Additional reagents and equipment for counting cells ( appendix 3A; Strober, )

Support Protocol 1: Metal Content Determination for In‐House Antibody Conjugates

  Materials
  • In‐house metal conjugated antibody
  • Digestion solution: 2% (v/v) solution in Mill‐Q water of BASELINE hydrochloric acid (Seastar Chemicals Inc., cat. no. S020401) or equivalent containing less than 1 ppb metal content
  • Dilution solution: 2% (v/v) solution in Mill‐Q water of BASELINE nitric acid (Seastar Chemicals Inc., cat. no. S020101) or equivalent containing less than 1 ppb metal content
  • Tuning solution (Fluidigm, cat. no. 201072)
  • Wash solution (Fluidigm, cat. no. 201070)
  • Maxpar water (Fluidigm, cat. no. 201069)
  • 5‐ml round‐bottom polystyrene test tubes (Corning, cat. no. 352008)
  • Mass cytometer (Helios from Fluidigm)
  • CyTOF software, version 6.5.358
CAUTION: When handling hydrochloric or nitric acid, use personal protective equipment, as these acids are extremely corrosive and can cause chemical burns.CAUTION: Always add acid to water and not the other way, around to avoid explosion.

Basic Protocol 2: 33‐Parameter Human Immunophenotyping Panel with Cytoplasmic Targets

  Materials
  • PBMCs, frozen ( protocol 1)
  • Wash medium: RPMI 1640 (Gibco, cat. no. 11875085) containing 10% fetal bovine serum (FBS; Gibco, cat. no. 16140071) pre‐warmed at 37°C
  • Stimulation cocktail (see recipe)
  • Maxpar metal‐conjugated and validated antibodies (Tables 5.11.1 and 5.11.2)
  • Maxpar Fix I buffer (Fluidigm, cat. no. 201067): dilute 1:5 in PBS for working (1×) concentration
  • Maxpar Perm‐S buffer (Fluidigm, cat. no. 201066)
  • Maxpar Fix and Perm buffer (Fluidigm, cat. no. 201067)
  • Maxpar cell staining buffer (Fluidigm, cat. no. 201068)
  • 24‐well cell culture plate
  • Additional reagents and equipment for preparing immunophenotyping panel ( protocol 1)

Alternate Protocol 1: 33‐Parameter Human Immunophenotyping Panel with Cytoplasmic and Nuclear Targets

  Additional Materials (also see Basic Protocols protocol 11 and protocol 32)
  • Fix/Perm buffer: mix 1 part Fixation/Permeabilization Concentrate (eBiosciences, cat. no. 00‐5123‐43) with 3 parts Fixation/Permeabilization Diluent (eBiosciences, cat. no. 00‐5223‐56); dilute to 1× with Maxpar water (Fluidigm, cat. no. 201069)
  • 10× Permeabilization Buffer (eBiosciences, cat. no. 00‐8333‐56)
  • Maxpar metal‐conjugated and validated antibodies (Tables 5.11.1, 5.11.2, and 5.11.3)

Basic Protocol 3: Titration of Mass Cytometry Barcoding Reagents

  Materials
  • 50 million freeze/thawed human PBMCs
  • Cell fixative (1.6% paraformaldehyde working solution): dilute 16% paraformaldehyde (Electron Microscopy Sciences, cat. no. 15710) 1:10 in PBS (Gibco, cat. no. 10010023) for 1.6% working concentration
  • Maxpar cell staining buffer (Fluidigm, cat. no. 201068)
  • Cell‐IDTM 20‐Plex Pd Barcoding Kit (Fluidigm, cat. no. 201060)
  • Fluidigm debarcoder software or Nolan lab single‐cell debarcoder (Zunder et al., )
  • Additional reagents and equipment for preparing immunophenotyping panel ( protocol 1)
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Figures

Videos

Literature Cited

Literature Cited
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