Isolation of Human Monocyte Populations

Larry M. Wahl1, Sharon M. Wahl1, Lesley E. Smythies2, Phillip D. Smith2

1 National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, 2 University of Alabama at Birmingham, Birmingham, Alabama
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.6A
DOI:  10.1002/0471142735.im0706as70
Online Posting Date:  January, 2006
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Abstract

This unit describes the isolation of monocytes from lymphocytes by adherence, gradient sedimentation on colloidal silica particles, and flow cytometry. Because the first two methods can result in cell activation (induction of gene expression or protein secretion), and the third is technically difficult, a fourth protocol is presented which describes counterflow centrifugal elutriation. This latter procedure can be used to isolate large numbers of purified, nonactivated monocytes.

     
 
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Table of Contents

  • Basic Protocol 1: Isolation of Monocytes by Adherence
  • Basic Protocol 2: Isolation of Monocytes by Size Sedimentation on Percoll
  • Basic Protocol 3: Isolation of Monocytes by Flow Sorting
  • Basic Protocol 4: Isolation of Monocytes by Counterflow Centrifugal Elutriation
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Monocytes by Adherence

  Materials
  • Peripheral blood mononuclear cells (PBMCs; purified by Ficoll‐Hypaque sedimentation, unit 7.1)
  • Serum‐free DMEM medium (e.g., Invitrogen) supplemented with 2 mM L‐glutamine and 50 µg/ml gentamicin
  • 0.02% (w/v) disodium EDTA in phosphate‐buffered saline (PBS, appendix 2A; low pyrogen)
  • 75‐cm2 tissue culture flasks (Falcon)
  • Plastic cell scraper (optional)
  • Beckman GPR centrifuge with GH‐3.7 horizontal rotor (or equivalent)
  • 15‐ml conical polypropylene centrifuge tube
  • Additional reagents and equipment for cell counting ( appendix 3A)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and aseptic technique should be used accordingly.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified.

Basic Protocol 2: Isolation of Monocytes by Size Sedimentation on Percoll

  Materials
  • Fetal bovine serum (FBS; heat‐inactivated 1 hr, 56°C; also see appendix 2A)
  • Peripheral blood mononuclear cells (PBMCs; purified by Ficoll‐Hypaque centrifugation, unit 7.1)
  • Hanks' balanced salt solution (HBSS; appendix 2A) containing 10% (v/v) FBS
  • Percoll (specific gravity 1.130 g/ml, 17 mOs/kg water; Amersham Biosciences)
  • 2× phosphate‐buffered saline (PBS; see appendix 2A for 1×; also available premixed from Invitrogen)
  • 15‐ml conical polypropylene centrifuge tubes
  • Sorvall RC2‐B centrifuge with SS‐34 fixed‐angle rotor (or equivalent)
  • 15‐ml polycarbonate centrifuge tubes (Sorvall)
  • Beckman GPR centrifuge with GH‐3.7 horizontal rotor (or equivalent temperature‐controlled centrifuge)
  • Additional reagents and equipment for cell counting ( appendix 3A)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 3: Isolation of Monocytes by Flow Sorting

  Materials
  • Peripheral blood mononuclear cells (PBMCs; purified by Ficoll‐Hypaque gradient, unit 7.1)
  • Phosphate‐buffered saline (PBS; appendix 2A) without and with 10% (v/v) AB serum (heat‐inactivated 1 hr, 56°C; also see appendix 2A)
  • 10 µg/ml monocyte‐specific MAb (e.g., CD14; R&D Systems) in serum/azide solution
  • Serum/azide solution: 0.1% (w/v) NaN 3 and 2% (v/v) FBS in PBS for FACS analysis; omit azide for functional studies
  • Serum‐free DMEM (e.g., Invitrogen) supplemented with 2 mM L‐glutamine and 50 µg/ml gentamycin
  • Beckman GPR centrifuge with GH‐3.7 horizontal rotor (or equivalent)
  • Flow cytometer with sorting capabilities (e.g., FACS series, Becton Dickinson)
  • Additional reagents and equipment for immunofluorescent staining (unit 5.3) and flow cytometry/cell sorting (units 5.4 & 7.9)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 4: Isolation of Monocytes by Counterflow Centrifugal Elutriation

  Materials
  • 70% ethanol
  • Peripheral blood mononuclear cells (PBMCs; purified by Ficoll‐Hypaque gradient, unit 7.1)
  • Phosphate‐buffered saline (PBS; appendix 2A; low pyrogen)
  • Serum‐free DMEM (e.g., Invitrogen) supplemented with 2 mM L‐glutamine and 50 µg/ml gentamycin
  • 50% bleach
  • Beckman elutriation system (see Fig. ): J‐6M elutriation centrifuge with JE‐6B rotor, strobe, and standard elutriation chamber(s)
  • 2‐ml pipets
  • Silastic tubing, ¼ × 118–in.
  • Masterflex pump (Cole Parmer, 1 to 100 rpm, 10‐turn potentiometer, 115 V AC; with 7014 pump head, Cole‐Parmer cat. no. N‐07530‐35, and silicone tubing, Cole‐Parmer cat no. F6411‐14)
  • Ring stand and holder
  • Beckman GPR centrifuge with GH‐3.7 horizontal rotor (or equivalent)
  • Coulter counter with Channelyzer (Beckman Coulter)
  • 10‐ml pipet or 10‐ml syringe
NOTE: All solutions and equipment coming into contact with cells must be sterile, and aseptic technique should be used accordingly.
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Figures

  •   FigureFigure 7.6.1 Design of counterflow centrifugal elutriation system.
  •   FigureFigure 7.6.2 Configuration of sample reservoir (A) during loading of cells and (B) during elutriation.
  •   FigureFigure 7.6.3 Blood monocyte morphology and flow cytometric analysis for purity. Insets show postive control staining for CD3, CD20, and CD69 using peripheral blood mononuclear cells, and for CD83 using monocyte‐derived dendritic cells.

Videos

Literature Cited

Literature Cited
   Edelson, P.J. and Cohn, Z.A. 1976. Purification and cultivation of monocytes and macrophages. In In Vitro Methods in Cell‐Mediated and Tumor Immunity (B.A. Bloom and J.R. David, eds.) pp. 333‐340. Academic Press, San Diego.
   Fuhlbrigge, R.C., Chaplin, D.D., Kiely, J.M., and Unanue, E.R. 1987. Regulation of interleukin 1 gene expression by adherence and lipopolysaccharide. J. Immunol. 138:3799‐3802.
   Gmelig‐Meyling, F. and Waldmann, T.A. 1980. Separation of human blood monocytes and lymphocytes on a continuous percoll gradient. J. Immunol. Methods 33:1‐9.
   Haskill, S., Johnson, C., Eierman, D., Becker, S., and Warren, K. 1988. Adherence induces selective mRNA expression of monocyte mediators and proto‐oncogenes. J. Immunol. 140:1690‐1694.
   Heppelston, A.G. and Styles, J.A. 1967. Activity of a macrophage factor in collagen formation by silica. Nature 214:521‐522.
   Smith, P.D., Smythies, L.E., and Wahl, S.M. 2001. Macrophage effector function. In Clinical Immunology: Principles and Practice (R.R. Rich, T.A. Fleisher, W.T. Shearer, B.I. Kotzin, and H.W. Schroeder, Jr., eds.) pp. 99.1‐19.9. Harcourt Health Sciences, London.
   Smythies, L.E., Sellers, M., Clements, R.H., Mosteller‐Barnum, M., Meng, G., Benjamin, W.H., Orenstein, J.M., and Smith, P.D. 2005. Human intestinal macrophages display profound inflammatory anergy despite avid phagocytic and bacteriocidal acitivity. J. Clin. Invest. 115:66‐75.
   Wahl, L.M., Katona, I.M., Wilder, R.L., Winter, C.C., Haraoui, B., Scher, I., and Wahl, S.M. 1984. Isolation of human mononuclear cell subsets by counterflow centrifugal elutriation (CCE). I. Characterization of B‐lymphocyte‐, T‐lymphocyte‐, and monocyte‐enriched fractions. Cell. Immunol. 85:373‐383.
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