Isolation of Whole Mononuclear Cells from Peripheral Blood and Cord Blood

Ivan J. Fuss1, Marjorie E. Kanof2, Phillip D. Smith3, Heddy Zola4

1 Mucosal Immunity Section, National Institute of Health, Bethesda, Maryland, 2 GAO, U.S. Government Accountability Office, Department of Health and Human Services, Washington, D.C., 3 National Institute of Dental Research/NIH, Bethesda, Maryland, 4 Child Health Research Institute, North Adelaide, Australia
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.1
DOI:  10.1002/0471142735.im0701s85
Online Posting Date:  April, 2009
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Abstract

Peripheral blood is the primary source of lymphoid cells for investigation of the human immune system. Its use is facilitated by Ficoll‐Hypaque density gradient centrifugation—a simple and rapid method of purifying peripheral blood mononuclear cells (PBMC) that takes advantage of the density differences between mononuclear cells and other elements found in the blood sample. Thus, cells are distributed in the solution in layers based on the differences in their density/size. Additional purification methods can be employed as the mononuclear cell sample can be purified from monocytes by adherence or by exposure to L‐leucine methyl ester; these methods are described for both procedures. Cord blood and peripheral blood from infants contain immature cells, including nucleated red cells, which can result in significant contamination of the mononuclear cell layer, and removal of these cells requires additional steps that are described. The isolation procedures presented here can also be applied to cell populations derived from tissues. Curr. Protoc. Immunol. 85:7.1.1‐7.1.8. © 2009 by John Wiley & Sons, Inc.

Keywords: mononuclear cell; Ficoll‐Hypaque; density gradient; peripheral blood

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Mononuclear Cells by Ficoll‐Hypaque Gradient Centrifugation
  • Support Protocol 1: Depletion of Monocytes/Macrophages from Mononuclear Cells Using Adherence Method
  • Support Protocol 2: Depletion of Monocytes/Macrophages from Mononuclear Cells Using L‐Leucine Methyl Ester
  • Support Protocol 3: Depletion of Contaminating Cells from Mononuclear Cell Fractions from Cord or Infant Peripheral Blood
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of Mononuclear Cells by Ficoll‐Hypaque Gradient Centrifugation

  Materials
  • Heparinized blood ( appendix 3F) or heparinized cord blood
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Ficoll‐Hypaque solution (density 1.077 g/liter; see recipe)
  • Hanks' balanced salt solution (HBSS; appendix 2A)
  • Fetal bovine serum (FBS; e.g., HyClone), with heat inactivation (1 hr, 56°C; appendix 2A)
  • Complete RPMI‐1640 medium ( appendix 2A)
  • Pipets, sterile
  • 15‐ or 50‐ml conical centrifuge tubes
  • Beckman GPR centrifuge with GH‐3.7 horizontal rotor (or equivalent temperature‐controlled centrifuge)
  • Additional reagents and equipment necessary for counting cells and trypan blue exclusion for determining viability ( appendix 3A& ), and depletion of contaminating cells from mononuclear fractions (see protocol 4; optional)

Support Protocol 1: Depletion of Monocytes/Macrophages from Mononuclear Cells Using Adherence Method

  • Mononuclear cell suspension (see protocol 1)
  • Complete RPMI‐1640 medium ( appendix 2A), room temperature
  • 150‐cm2 tissue culture flasks, slanted‐neck
  • 37°C, 5% CO 2 humidified incubator

Support Protocol 2: Depletion of Monocytes/Macrophages from Mononuclear Cells Using L‐Leucine Methyl Ester

  • Mononuclear cell suspension (see protocol 1)
  • 1 mM L‐leucine methyl ester (Sigma) in complete RPMI medium (serum‐free and filter sterilized; appendix 2A)
  • Additional reagents and equipment for nonspecific esterase staining ( appendix 3C) or for flow cytometry (unit 7.9)

Support Protocol 3: Depletion of Contaminating Cells from Mononuclear Cell Fractions from Cord or Infant Peripheral Blood

  • Mononuclear cell suspension (see protocol 1)
  • ACK lysing buffer (unit 3.1)
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Figures

  •   FigureFigure 7.1.1 Ficoll‐Hypaque density isolation of mononuclear cells. Ficoll‐Hypaque is underlayered onto whole blood or cord blood. The sample is centrifuged allowing the separation of lymphocyte cell population from other blood elements based upon density gradient.
  •   FigureFigure 7.1.2 Separation of blood components on a Ficoll‐Hypaque gradient.
  •   FigureFigure 7.1.3 Separation of adult (A) and cord (B) blood under identical conditions. The cord blood sample shows significant contamination of the interface layer with red blood cells.

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Literature Cited

Literature Cited
   Boyum, A. 1968. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Invest. Suppl. 21:77‐89.
   Ferrante, A. and Thong, Y. 1980. Optimal conditions for simultaneous purification of mononuclear and polymorphonuclear leucocytes from human blood by the Hypaque‐Ficoll method. J. Immunol. Methods 36:109.
   Kaplan, J., Nolan, D., and Ree, A. 1982. Altered lymphocyte markers and blastogenic response associated with 24 hour delay in processing of blood samples. J. Immunol. Methods 50:187‐191.
   Niku, S., Hoon, D., Cochran, A., and Morton, D. 1987. Isolation of lymphocytes from clotted blood. J. Immunol. Methods. 105:9‐14.
   Nisperos, B. 1990. Density gradient isolation of peripheral blood lymphocytes. In ASHI Laboratory manual, 2nd ed. (A.A. Zachary and G.A. Teresi, eds.) pp. 23‐27. American Society for Histocompatibility and Immunogenics, Lenexa, Kans.
   Ridings, J., Weedon, H., Ioannu, C., Flego, L., Macadle, P., and Zola, H. 1996. Purification of cord blood lymphocytes. J. Immunol. Methods. 195:43‐48.
   Skoog, W. and Beck, W. 1956. Studies of the fibrinogen, dextran and phytohaemagglutinin methods of isolating leukocytes. Blood 11:436.
   Tamul, K., Schmitz, J., Kane, K., and Folds, J. 1995. Comparison of the effects of Ficoll‐Hypaque separation and whole blood lysis on results of immunophenotypic analysis of blood and bone marrow samples from patients with hematologic malignancies. Clin. Diag. Lab. Immunol. 2:337‐342.
   Thiele, D., Kurosaka, M., and Lipsky, P. 1983. Phenotype of the accessory cell necessary for mitogen‐stimulated T and B cell responses in human peripheral blood: Delineation by its sensitivity to the lysosomotropic agent, L‐leucine methyl ester. J. Immunol. 131:2282‐2290.
   Thong, Y., Currell, J., and Rodwell, R. 1983. The rapid one‐step gradient centrifugation procedure for simultaneous isolation of granulocytic and mononuclear leukocytes from human blood: biological, physical and chemical bases. Med. Hypotheses 12:103‐111.
   Tse, H. and Dutton, R. 1976. Separation of helper and suppressor T lymphocytes on a Ficoll velocity sedimentation gradient. J. of Exp. Med. 143:1199‐1210.
   Vissers, M., Jester, S., and Fantone, J. 1988. Rapid purification of human peripheral blood monocytes by centrifugation through Ficoll‐Hypaque and Sepracell‐MN. J. Immunol. Methods 13:203‐207.
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