Preparation and Culture of Human Lymphocytes

William E. Biddison1

1 National Institute of Neurological Disorders and Stroke/NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 2.2
DOI:  10.1002/0471143030.cb0202s00
Online Posting Date:  May, 2001
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Abstract

This unit presents protocols for preparation of lymphocytes from peripheral (whole) blood or leucopherisis samples, first by differential centrifugation in Ficoll‐Hypaque to separate them from erythrocytes and granulocytes. Repeated centrifugation removes the plasma and platelets. Monocyte/macrophage cells and dendritic cells can be purified from the T and B cell population by exploiting their adhesion to serum‐coated plates in the presence of recombinant IL‐3. Different subpopulations of lymphocytes can be isolated by specific antibodies bound to magnetic beads.

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Lymphocytes by Ficoll‐Hypaque Gradient Centrifugation
  • Basic Protocol 2: Preparation of Monocytes/Macrophages and “Dendritic‐like” Cells from Lymphocyte Populations
  • Basic Protocol 3: Positive Selection of T and B Cells by Monoclonal Antibody–Coated Magnetic Beads
  • Alternate Protocol 1: Positive Selection of T and B Cells by Monoclonal Antibodies and Anti‐IgG‐Coated Magnetic Beads
  • Alternate Protocol 2: Isolation of T and B Cell Subpopulations by Negative Selection
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Lymphocytes by Ficoll‐Hypaque Gradient Centrifugation

  Materials
  • Anticoagulated whole blood or white blood cells from leukapheresis donor
  • Phosphate‐buffered saline (PBS), without calcium or magnesium (Bio‐Whittaker), room temperature
  • Ficoll‐Hypaque solution (see recipe), room temperature
  • Lymphocyte culture medium (LCM; see recipe), room temperature
  • Iscove's modified Dulbecco's medium (IMDM; Life Technologies) containing 20% heparinized human plasma
  • Freezing medium (see recipe)
  • 50‐ml conical centrifuge tubes
  • Sorvall RT‐6000B centrifuge with H‐1000 rotor (or equivalent)
  • 1.5‐ml cryotubes (e.g., Nunc)
  • Controlled‐rate freezer (e.g., CryoMed from Forma Scientific)
  • Liquid nitrogen freezer
  • Additional reagents and equipment for counting cells and determining cell viability (unit 1.1) and flow cytometry (Robinson et al., )

Basic Protocol 2: Preparation of Monocytes/Macrophages and “Dendritic‐like” Cells from Lymphocyte Populations

  Materials
  • Lymphocyte culture medium (LCM; see recipe), 37°C
  • Lymphocyte population (see protocol 1)
  • Recombinant human interleukin 3 (rhIL‐3), interleukin 4 (rhIL‐4), and GM‐CSF (PeproTech)
  • 5 mM tetrasodium EDTA in PBS (also available as Versene from Life Technologies), filter‐sterilized using 0.22‐µm Nalgene filter, prewarmed to 37°C
  • 175‐cm2 tissue culture flasks
  • 50‐ml conical centrifuge tubes
  • Sorvall RT‐6000B centrifuge with H‐1000 rotor (or equivalent)
  • Additional reagents and equipment for counting cells and determining cell viability (unit 1.1) and flow cytometry (Robinson et al., )

Basic Protocol 3: Positive Selection of T and B Cells by Monoclonal Antibody–Coated Magnetic Beads

  Materials
  • Lymphocyte population (see protocol 1)
  • Anti‐CD3 and anti‐CD19 antibodies for flow cytometry (Becton‐Dickinson or Coulter)
  • Anti‐CD3‐ and anti‐CD19‐coated magnetic beads (Dynabeads M‐450; Dynal)
  • PBS without calcium and magnesium (Bio‐Whittaker)
  • PBS/HSA: PBS without calcium and magnesium (Bio‐Whittaker) containing 0.5% (w/v) human serum albumin (American Red Cross Blood Services)
  • IMDM/HSA: Iscove's modified Dulbecco's medium (Life Technologies) containing 0.5% (w/v) human serum albumin
  • Polyclonal anti–mouse Fab antiserum (Detachabead; Dynal)

Alternate Protocol 1: Positive Selection of T and B Cells by Monoclonal Antibodies and Anti‐IgG‐Coated Magnetic Beads

  • Anti‐CD3 and anti‐CD19 IgG monoclonal antibodies (Becton Dickinson or Coulter)
  • Goat anti‐mouse IgG–coated magnetic beads (Dynabeads M‐450; Dynal)

Alternate Protocol 2: Isolation of T and B Cell Subpopulations by Negative Selection

  • Anti‐CD3, anti‐CD14, anti‐CD16, and anti‐CD19 IgG monoclonal antibodies (Becton‐Dickinson or Coulter)
  • Goat anti‐mouse IgG–coated magnetic beads (Dynabeads M‐450; Dynal)
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Figures

Videos

Literature Cited

   Biddison, W., Taub, D., Cruikshank, W., Center, D., Connor, E., and Honma, K. 1997. Chemokine and matrix metalloproteinase secretion by myelin proteolipid protein–specific CD8+ T cells. J. Immunol. 158:3046‐3053.
   Boyum, A. 1968. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Invest. 21(Suppl. 97):107‐111.
   Funderud, S., Erikstein, B., Asheim, H., Nustad, K., Stokke, T., Blomhoff, H., Holte, H., and Smeland, E. 1990. Functional properties of CD19+ B lymphocytes positively selected from buffy coats by immunomagnetic separation. Eur. J. Immunol. 20:201‐206.
   Lea, T., Smeland, E., Funderud, S., Vartdal, F., Davies, C., Beiske, K., and Ugelstad, J. 1986. Characterization of human mononuclear cells after positive selection with immunomagnetic particles. J.Immunol. 23:509‐519.
   Robinson, J.P., Darzynkiewicz, Z., Dean, P.N., Orfan, A., Rabinovitch, P.S., Stewart, C.C., Tanke, H.J., and Wheeless, L.L. 1998. Current Protocols in Cytometry. John Wiley & Sons, New York.
   Sallusto, F. and Lanzavecchia, A. 1994. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony‐stimulating factor plus interleukin 4 and down‐regulated by tumor necrosis factor alpha. J. Exp. Med. 179:1109‐1118.
   Vartdal, F., Gaudernack, G., Ugelstad, J., Kvalhelm, G., Lea, T., Bosnes, V., and Albrechtsen, D. 1987. Depletion of T lymphocytes from human bone marrow. Use of magnetic monosized polymer microspheres coated with T lymphocyte specific monoclonal antibodies. Transplantation 43:366‐371.
Key References
   Boyum, 1968. See above.
  Comprehensive explanation of procedure for Ficoll‐Hypaque gradient separation of human lymphocytes.
   Funderud, S., Nustad, K., Lea, T., Vartdal, F., Gaudernack, G., Stensted, P., and Ugelstad, J. 1987. Fractionation of lymphocytes by immunomagnetic beads. In Lymphocytes: A Practical Approach (G.G.B. Klaus, ed.) pp. 55‐61. Oxford University Press, New York.
  Comprehensive description of magnetic bead technology for lymphocyte separations.
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