Preparing Viable Single Cells from Human Tissue and Tumors for Cytomic Analysis

Nalin Leelatian1, Deon B. Doxie1, Allison R. Greenplate2, Justine Sinnaeve1, Rebecca A. Ihrie3, Jonathan M. Irish2

1 Department of Cancer Biology, Vanderbilt University, Nashville, Tennessee, 2 Department of Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, Tennessee, 3 Department of Cell and Developmental Biology, Vanderbilt University, Nashville, Tennessee
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 25C.1
DOI:  10.1002/cpmb.37
Online Posting Date:  April, 2017
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Abstract

Mass cytometry is a single‐cell biology technique that samples >500 cells per second, measures >35 features per cell, and is sensitive across a dynamic range of >104 relative intensity units per feature. This combination of technical assets has powered a series of recent cytomic studies where investigators used mass cytometry to measure protein and phospho‐protein expression in millions of cells, characterize rare cell types in healthy and diseased tissues, and reveal novel, unexpected cells. However, these advances largely occurred in studies of blood, lymphoid tissues, and bone marrow, since the cells in these tissues are readily obtained in single‐cell suspensions. This unit establishes a primer for single‐cell analysis of solid tumors and tissues, and has been tested with mass cytometry. The cells obtained from these protocols can be fixed for study, cryopreserved for long‐term storage, or perturbed ex vivo to dissect responses to stimuli and inhibitors. © 2017 by John Wiley & Sons, Inc.

Keywords: human tissue; human tumor; dissociation; single cell; cytometry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Viable Single Cells from Human Tissue and Tumors
  • Basic Protocol 2: Preparation of Cells for Mass Cytometry
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Viable Single Cells from Human Tissue and Tumors

  Materials
  • Tissue sample
  • Phosphate‐buffered saline (PBS; Corning/Mediatech, cat. no. 21040CV,), room temperature
Experimental media:
  • For glioma: DMEM/F12 + GlutaMax (Gibco/Life Technologies, cat. no. 10565018) with a defined hormone and salt mix (Reynolds, Tetzlaff, & Weiss, ) and 50 µg/ml gentamicin sulfate (Corning/Mediatech, cat. no. 30‐005‐CR)
  • For melanoma: MEM (Corning/Mediatech, cat. no. 10010CV) with 10% FBS (Thermo Fisher Scientific, cat. no. 26140079) and 100 U/ml penicillin/100 µg/ml streptomycin (add from 100× penicillin‐streptomycin solution, GE Healthcare, cat. no. SV30010)
  • For tonsils: RPMI 1640 (Corning/Mediatech, cat. no. 10040CV) with 10% FBS (Thermo Fisher Scientific, cat. no. 26140079) and 100 U/ml penicillin/100 µg/ml streptomycin (add from 100× penicillin‐streptomycin solution, GE Healthcare, cat. no. SV30010)
  • 20× collagenase II: dilute collagenase from Clostridium histolyticum (Sigma, cat. no. C6885) to 2500 CDU/ml (20 mg/ml) in PBS (store at –80ºC)
  • 100× DNase I: dilute DNase I from bovine pancreas (Sigma‐Aldrich, cat. no. DN25) to 10,000 Kunitz Units/ml in PBS (store at –80ºC)
  • ACK lysing buffer (Lonza, cat. no. 10‐548E)
  • Trypan blue (Hyclone, cat. no. SV30084.01, prepared as recommended by manufacturer; also see appendix 3F; Phelan & May, )
  • DMSO (Catalog no. BP231‐1, Fisher Scientific, MA)
  • 15‐ml (Corning Falcon, cat. no. 430055) and 50‐ml (Corning Falcon, cat. no. 430829) conical tubes
  • Benchtop centrifuge with swing‐out rotor (Sorvall model ST 16; Thermo Scientific)
  • 60‐mm petri dish (Fisher Scientific, cat. no. FB0875713)
  • P1000 plastic pipet tips with narrow end cut to make a wide opening (diameter ∼2 to 3 mm)
  • Scalpels with blade no.10 (Fisher Scientific, cat. no. 12‐460‐451)
  • Incubator set at 37°C, 5% CO 2
  • Nutating platform placed inside incubator, set to 18 rpm (Fisher Scientific, cat. no. 05‐450‐213)
  • 70‐µm (Corning Falcon, cat. no. 431751) and 40‐µm (Corning Falcon, cat. no. 431750) cell strainers sized to fit 50‐ml conical tubes
  • Inverted phase contrast microscope for cell culture (use 10× objective magnification for quantifying cell viability)
  • 1.8‐ml cryogenic tubes with cap (Thermo Fisher Scientific, cat. no. 377267)
  • Additional reagents and equipment for counting viable cells by trypan blue exclusion ( appendix 3F; Phelan & May, )

Basic Protocol 2: Preparation of Cells for Mass Cytometry

  Materials
  • Perm 1: room‐temperature 0.02% (w/v) saponin (Calbiochem, cat. no. 558255) in PBS
  • Perm 2: ice‐cold 100% methanol (Fisher Scientific, cat. no. A412‐4) kept at −20°C until immediately prior to adding to cells
  • Dissociated single cells ( protocol 1)
  • Experimental media:
  • For glioma: DMEM/F12 plus GlutaMax (Gibco/Life Technologies, cat. no. 10565018) with a defined hormone and salt mix (Reynolds, Tetzlaff, & Weiss, ) and 50 µg/ml gentamicin sulfate (Corning/Mediatech, cat. no. 30‐005‐CR)
  • For melanoma: MEM (Corning/Mediatech, cat. no. 10010CV) with 10% FBS (Thermo Fisher Scientific, cat. no. 26140079) and 100 U/ml penicillin/100 µg/ml streptomycin (add from 100× penicillin‐streptomycin stock solution, GE Healthcare, cat. no. SV30010)
  • For tonsils: RPMI 1640 (Corning/Mediatech, cat. no. 10040CV) with 10% FBS (Thermo Fisher Scientific, cat. no. 26140079) and 100 U/ml penicillin/100 µg/ml streptomycin (add from 100× penicillin‐streptomycin stock solution; GE Healthcare, cat. no. SV30010)
  • Optional: 100× DNase I: dilute DNase I from bovine pancreas (Sigma‐Aldrich, cat. no. DN25) to 10,000 Kunitz Units/ml in PBS (store at –80ºC)
  • Staining medium: 1% (w/v) bovine serum albumin (BSA; Fisher Scientific, cat. no. BP9703100) in PBS
  • Live Stain reagent mix: A combined solution of all relevant antibodies (antibody list in Table 25.1.1)
  • Phosphate‐buffered saline (PBS; Corning/Mediatech, cat. no. 21040CV)
  • 16% paraformaldehyde (PFA; Electron Microscopy Sciences, cat. no. 15710)
  • Saponin Stain reagent mix: A combined solution of all relevant antibodies (antibody list in Table 25.1.1)
  • Methanol Stain reagent mix: A combined solution of all relevant antibodies (antibody list in Table 25.1.1)
  • 1× Four Elements Calibration Beads (Fluidigm, cat. no. 201078)
  • 15‐ml conical tubes (Corning Falcon, cat. no. 430055)
  • Benchtop centrifuge with swing‐out rotor (Sorvall model ST 16; Thermo Scientific)
  • 5‐ml round‐bottom FACS tubes without cap (Corning Falcon, cat. no. 352052)
  • Rotor adapters with round buckets that accommodate 5 ml FACS tubes (Thermo Fisher Scientifc, cat. no. 75003680)
  • 5‐ml round‐bottom FACS tubes with filter caps (Corning Falcon, cat. no. 352235)
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Figures

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Literature Cited

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