Immunomagnetic Purification of T Cell Subpopulations

Kevin Horgan1, Stephen Shaw1, Monica Boirivant2

1 National Cancer Institute, Bethesda, Maryland, 2 Instituto Superiore di Sanità, Viale Regina Elena, Rome, Italy
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.4
DOI:  10.1002/0471142735.im0704s85
Online Posting Date:  April, 2009
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Abstract

There are two types of magnetic cell isolation technologies, one column‐based and the other tube‐based. The column‐based technology utilizes nano‐sized particles that need to pass through a ferromagnetic spheres column to increase cell‐capture capacity. The tube‐based system utilizes micron‐sized beads that can be selected using a magnet applied to the tube. The beads are used for direct or indirect labeling of cells. Direct labeling is achieved with antibodies coupled to magnetic particles directly added to the cell suspension. For indirect labeling the cells are first labeled with the antibody of interest; the antibody can be simple, biotinylated, or fluorochrome‐conjugated. Subsequently, beads coated with streptavidin or anti‐immunoglobulin, anti‐biotin, anti‐fluorochrome antibodies are used to specifically mark the subpopulation of interest. Separation of target cells can be achieved using positive or negative selection or a combination of the two. Quality of the sample preparation is critical to obtain good purification and yield. Curr. Protoc. Immunol. 85:7.4.1‐7.4.9. © 2009 by John Wiley & Sons, Inc.

Keywords: immunomagnetic purification; T cell subpopulations; beads; magnet; antibodies

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Immunomagnetic Negative Selection of T Cells from Peripheral Blood Mononuclear Cells: Tube‐Based System
  • Alternate Protocol 1: Column‐Based Negative Selection of T Cells from Peripheral Blood Mononuclear Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Immunomagnetic Negative Selection of T Cells from Peripheral Blood Mononuclear Cells: Tube‐Based System

  Materials
  • Appropriate monoclonal antibodies (10× MAb mix including CD14, CD16, CD20, and anti‐glycophorin; see Table 7.4.1 and Reagents and Solutions)
  • Peripheral blood mononuclear cells after Ficoll‐Hypaque separation (PBMC; unit 7.1)
  • Coating medium/isolation buffer (see recipe), room temperature and 4°C, freshly prepared
  • Monoclonal human anti–mouse IgG–coated magnetic beads (Dynabeads Pan Mouse IgG, Invitrogen, cat. no. 110.41, 110.42) or goat anti–mouse IgG–coated magnetic beads (Dynabeads, Invitrogen, cat. no 110.33; see )
  • 15‐ml polypropylene tubes (e.g., Falcon, cat. no. 2059)
  • End‐over‐end rotator
  • Sorvall RT 6000 centrifuge with H‐1000B rotor (or equivalent)
  • Magnetic separation apparatus (DynaMag‐15, Invitrogen, cat. no. 123‐01D or DynaMag‐50, Invitrogen, cat. no. 123‐02D)
  • Additional reagents and equipment for preparation and titration of monoclonal antibodies (unit 2.5), counting cells ( appendix 3A), flow cytometry (units 5.4& 7.9), and cryopreservation of cells ( 3.NaN)
NOTE: A kit containing the Dynabeads coated with a monoclonal human anti–mouse IgG antibody, a mixture of mouse IgG antibodies for CD14, CD16 (CD16a and CD16b), CD19, CD36, CD56, CDw123, and CD235a (Glycophorin A) for T cell negative isolation is available from Invitrogen (Dynabeads Untouched Human T cells, cat. no. 113.44D). The kit can process up to 1 × 109 cells. The protocol outlined by the manufacturer is based on 1 × 107 MNC and it is scalable from 1 × 107 to 5 × 108 cells.NOTE: Perform all steps at 4°C on ice or in a cold room under sterile conditions. All resuspensions of cells and washing of magnetic beads are done using coating medium.
Table 7.4.1   MaterialsExamples of Antibodies Used to Label Cells to be Removed

Contaminating cell types Receptor a Monoclonal antibody and source b
Monocytes CD14 MMA (ATCC)
My4 (Coulter)
Leu‐M3 (Becton Dickinson)
63D3 (ATCC)
CD11b c LM2/1 (ATCC)
M1/70 (ATCC)
Natural killer CD16 Leu‐11 (Becton Dickinson)
RBC Anti‐glycophorin 10F7 (ATCC)
B cells CD20 B1 (Coulter)
Leu‐16 (Becton Dickinson)

 aAlso see Table A.4A.1 ( appendix 4A) for complete listing of CD molecules.
 bAddresses and phone numbers of suppliers are provided in appendix 55.
 cOptional, depending on the precise cell population desired.

Alternate Protocol 1: Column‐Based Negative Selection of T Cells from Peripheral Blood Mononuclear Cells

  • Appropriate monoclonal antibodies (10× MAb mix including CD14, CD16, CD20, and anti‐glycophorin; see Table 7.4.1 and Reagents and Solutions)
  • Goat anti–mouse IgG MicroBeads (Miltenyi, cat. no. 130‐048‐402)
  • 30‐µm mesh (preseparation filter, Miltenyi, cat. no. 130‐041‐407)
  • MACS column (LD or LS)
  • MACS separator (MidiMACS)
NOTE: A kit for negative isolation of T cells is available from Miltenyi Biotech. The Pan T Cell Isolation Kit II, human (cat. no. 130‐091‐156) is an indirect magnetic labeling system. Non‐T cells are incubated with a cocktail of biotin‐conjugated antibodies against CD14, CD16, CD19, CD36, CD56, CD123, and CD235a. These cells are subsequently labeled with Anti‐Biotin MicroBeads to be magnetically depleted by retention in a MACS column placed in a MACS Separator. T cells pass through the column and are collected as the unlabeled cell fraction.
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Figures

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Literature Cited

   Berry, C.C., Wells, S., Charles, S., Aitchison, G., and Curtis, A.S. 2004. Cell response to dextran‐derivatised iron oxide nanoparticles post internalisation. Biomaterials 25:5405‐5413.
   Neurauter, A.A., Bonyhadi, M., Lien, E., Nøkleby, L., Ruud, E., Camacho, S., and Aarvak, T. 2007. Cell isolation and Expansion Using Dynabeads. Adv. Biochem. Engin./Biotechnol. 106:41‐73.
   Pisanic, T.R., II, Blackwell, J.D., Shubayev, V.I., Finones, R.R., and Jin, S. 2007. Nanotoxicity of iron oxide nanoparticle internalization in growing neurons. Biomaterials 28:2572‐2581.
Key Reference
   Neurauter, 2007. See above.
  Comprehensive description of magnetic bead technique.
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