Isolation of Human B Cell Populations

Guido Heine1, Gary P. Sims2, Margitta Worm3, Peter E. Lipsky2, Andreas Radbruch1

1 Deutsches Rheuma‐Forschungszentrum Berlin, Berlin, Germany, 2 National Institutes of Health, Bethesda, Maryland, 3 Klinik für Dermatologie, Venerologie und Allergologie, Charité ‐ Universitätsmedizin Berlin, Berlin, Germany
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.5
DOI:  10.1002/0471142735.im0705s94
Online Posting Date:  August, 2011
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Abstract

To study the function and biology of human B cells, it is necessary to isolate pure populations. Historically, B cells were enriched by the sequential depletion of monocytes, natural killer cells, and T cells. However, this time‐consuming process has been superseded by sorting methods using specific antibodies, targeting, in negative‐selection strategies, unwanted cell types, or, in positive‐selection strategies, B cell markers such as CD19. Here we describe in detail four methods for isolating B cells from human blood or mononuclear cells, and discuss how these techniques can be combined with fluorescent cell sorting for the characterization of specific B cell populations. Curr. Protoc. Immunol. 94:7.5.1‐7.5.14. © 2011 by John Wiley & Sons, Inc.

Keywords: B cell; CD19; CD43; purification; isolation; negative selection; magnetic separation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of B Cells Directly from Whole Blood by Negative Selection
  • Basic Protocol 2: Isolation of B Cells from Mononuclear Cells by Negative Selection Without Using a Column
  • Basic Protocol 3: Isolation of CD43 Negative B Cells by Column‐Based Magnetic Cell Sorting (MACS)
  • Basic Protocol 4: Immunomagnetic Isolation of Human B Cells by Positive Selection
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Isolation of B Cells Directly from Whole Blood by Negative Selection

  Materials
  • Anticoagulated human whole blood ( appendix 3F)
  • RosetteSep Human B Cell Enrichment Cocktail (registered trademark of StemCell Technologies; includes MAbs for human cell surface antigens CD2, CD3, CD16, CD56, CD66b, and glycophorin A)
  • Phosphate‐buffered saline (PBS; appendix 2A) containing 2% (v/v) fetal bovine serum (FBS, heat‐inactivated 1 hr at 56°C; appendix 2A), room temperature and ice cold
  • Ficoll‐Paque PLUS (Amersham Biosciences)
  • ACK lysing buffer (optional; see recipe; also available commercially, e.g., from BioSource)
  • Complete RPMI‐10 ( appendix 2A)
  • B cell–specific antibodies ( appendix 4A; see unit 5.3 for preparation)
  • 15‐ or 50‐ml centrifuge tubes (e.g., Corning)
  • Sorvall Heraeus centrifuge with no. 6434 rotor (or equivalent centrifuge with swinging‐bucket rotor)
  • Fine nylon mesh, sterile
  • Additional reagents and equipment for cell counting ( appendix 3A), immunofluorescence staining (unit 5.3), and flow cytometric analysis (unit 5.4)
NOTE: Unless otherwise noted, perform all steps at 4°C on ice or in cold room.

Basic Protocol 2: Isolation of B Cells from Mononuclear Cells by Negative Selection Without Using a Column

  Materials
  • Peripheral blood or tissue of interest
  • Phosphate‐buffered saline (PBS; appendix 2A) containing 2% (v/v) fetal bovine serum (FBS, heat‐inactivated 1 hr at 56°C; appendix 2A)
  • EasySep Negative Selection Human B Cell Enrichment Cocktail (registered trademark of StemCell Technologies; includes MAbs for human cell surface antigens CD2, CD3, CD14, CD16, CD36, CD56, and Glycophorin A, and for dextran on the nanoparticles)
  • EasySep Magnetic Nanoparticles (StemCell Technologies)
  • Complete RPMI‐10 ( appendix 2A)
  • B cell–specific antibodies ( appendix 4A; see unit 5.3 for preparation)
  • 12 × 75–mm polystyrene tubes (5‐ml tubes suitable for flow cytometry, e.g., Falcon)
  • EasySep Magnet (StemCell Technologies)
  • 15‐ml conical centrifuge tubes
  • Additional reagents and equipment for preparing mononuclear cells from peripheral blood (unit 7.1) or tissue (unit 7.8), counting cells ( appendix 3A), immunofluorescence staining (unit 5.3), and flow cytometric analysis (unit 5.4)
NOTE: Unless otherwise noted, perform all steps at 4°C on ice or in cold room.

Basic Protocol 3: Isolation of CD43 Negative B Cells by Column‐Based Magnetic Cell Sorting (MACS)

  Materials
  • Peripheral blood mononuclear cells (unit 7.1) or tissue cells (e.g., unit 7.8)
  • PBS/BSA: phosphate‐buffered saline (PBS; appendix 2A) supplemented with 0.5% (w/v) BSA
  • CD43, CD3, and CD14 magnetic microbeads (Miltenyi Biotec) or anti‐human CD43, anti‐human CD3, and anti‐CD14, available from BD Biosciences, all conjugated to phycoerythrin (PE; see unit 5.3, Support Protocol 4, for PE conjugation); and anti‐PE magnetic microbeads (Miltenyi Biotec)
  • Fluorochrome‐conjugated monoclonal antibodies against CD19 (B cells), CD14 (monocytes), and CD3 (T cell receptor) for flow cytometry
  • Propidium iodide (Sigma)
  • Refrigerated centrifuge
  • LS column (for ≤108 PBMCs per column) or XS column (for ≤109 PBMCs); both available from Miltenyi Biotec
  • Magnet (MidiMACS/SuperMACS) and stand (Miltenyi Biotec)
  • 26‐G needles
  • 15‐ml plastic tubes
  • Additional reagents and equipment for counting cells ( appendix 3A) and flow cytometry (units 5.3& 5.4)

Basic Protocol 4: Immunomagnetic Isolation of Human B Cells by Positive Selection

  Materials
  • Peripheral blood or tissue of interest, or cryopreserved ( appendix 3G) mononuclear cells
  • Phosphate‐buffered saline (PBS; appendix 2A) containing 2% (v/v) fetal bovine serum (FBS, heat‐inactivated 1 hr at 56°C; appendix 2A)
  • CD19 MicroBeads (Miltenyi Biotec)
  • Complete RPMI‐10 ( appendix 2A)
  • Biotinylated anti‐CD20 or anti‐CD19 antibody ( appendix 4A; see unit 5.3 for preparation/biotinylation), and streptavidin microbeads (Miltenyi Biotec)
  • MACS wash solution (see recipe), ice cold
  • Refrigerated centrifuge
  • LS columns and LS column adaptor (Miltenyi Biotec)
  • SuperMACS Separator (Miltenyi Biotec)
  • 15‐ml polypropylene conical tubes (e.g., Falcon)
  • Additional reagents and equipment for preparation of mononuclear cells from peripheral blood (unit 7.1) or tissue (unit 7.8), counting cells ( appendix 3A), immunofluorescence staining (unit 5.3), and flow cytometric analysis (unit 5.4)
NOTE: Unless otherwise noted, perform all steps at 4°C on ice or in cold room.
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Literature Cited

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Internet Resources
  http://www.stemcell.com
  StemCell Technologies Web site.
  http://www.miltenyi.com
  Miltenyi Biotec Web site.
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