Monocyte and Neutrophil Isolation and Migration Assays

Simon Yona1, Richard Hayhoe2, Inbal Avraham‐Davidi3

1 The Weizmann Institute of Science, Rehovot, Israel, 2 University of Cambridge, Cambridge, United Kingdom, 3 The Hebrew University–Hadassah Medical School, Jerusalem, Israel
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.15
DOI:  10.1002/0471142735.im1415s88
Online Posting Date:  February, 2010
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Abstract

This unit describes methods for isolating mouse monocytes and neutrophils, as well as in vitro protocols for measuring cell migration and polarization. The method employed here for the isolation of naïve phagocytes overcomes many of the difficulties previously encountered concerning phagocyte activation. Three in vitro protocols are provided for the analysis of cell migration, one requiring no specialized equipment, one requiring the modified Boyden chamber, and the other employing a flow chamber, which measures cell adhesion, rolling, and migration. Finally, a method is provided for imaging polarized cells by confocal microscopy. Curr. Protoc. Immunol. 88:14.15.1‐14.15.14. © 2010 by John Wiley & Sons, Inc.

Keywords: monocyte; neutrophil; chemotaxis; flow chamber; cell polarization

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Murine Bone Marrow Monocytes
  • Basic Protocol 2: Isolation of Murine Circulating Neutrophils
  • Basic Protocol 3: Chemotaxis Assay
  • Alternate Protocol 1: Measuring Phagocyte Migration Using a Flow Chamber
  • Support Protocol 1: Cell Polarization Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Murine Bone Marrow Monocytes

  Materials
  • Naive mice (typically 6 to 10 weeks old)
  • 70% ethanol
  • Phosphate‐buffered saline (PBS; Invitrogen)
  • Ficoll‐Paque Plus (GE Healthcare)
  • MACS buffer (see recipe)
  • Goat IgG (Sigma‐Aldrich)
  • Biotin‐conjugated anti‐CD115 (Clone AFS98; eBiosciences)
  • Streptavidin‐conjugated MACS beads (Miltenyi Biotec, cat. no. 130048101)
  • RPMI 1640 medium supplemented with 0.1% (w/v) bovine serum albumin (BSA)
  • Dissecting instruments:
    • Corkboard
    • Scissors
    • Forceps
  • 60‐mm petri dish
  • 15‐ and 50‐ml conical centrifuge tubes
  • 5‐ml syringe
  • 26‐G hypodermic needle
  • Refrigerated centrifuge
  • MACS LS Column (Miltenyi Biotec)
  • MidiMACS magnetic separator (Miltenyi Biotec)
  • Additional reagents and equipment for euthanasia of mice by carbon dioxide asphyxiation (unit 1.8) and counting cells using a hemacytometer ( appendix 3A)

Basic Protocol 2: Isolation of Murine Circulating Neutrophils

  Materials
  • Heparin solution (1000 U/ml)
  • Donor mice (typically 6 to 10 weeks old)
  • Dextran solution: 1.25 g dextran T500 (GE Healthcare) in 100 ml 0.9% NaCl
  • PBS‐BSA: 0.2 g bovine serum albumin (BSA) in 100 ml PBS
  • Phosphate‐buffered saline (PBS; Invitrogen)
  • Antibodies (Table 14.15.1):
    • Rat anti‐mouse CD2 (Clone RM2‐5; PharMingen)
    • Rat anti‐mouse CD5 (Clone 53.73; PharMingen)
    • Rat anti‐mouse CD45R B220 (Clone RA3‐6B2; PharMingen)
    • Rat anti‐mouse ICAM‐1 (Clone YN1/1.7.4; eBiosciences)
    • Rat anti‐mouse F4/80 (Clone F4/80; AbD Serotec, http://www.abdserotec.com)
  • Goat anti–rat IgG MicroBeads (Miltenyi Biotec)
  • 0.2% sodium chloride solution: 0.1 g sodium chloride in 50 ml distilled water
  • 1.6% sodium chloride solution: 0.8 g sodium chloride and 0.05 g glucose in 50 ml distilled water
  • RPMI 1640 medium supplemented with 0.1% (w/v) bovine serum albumin (BSA)
  • 1‐ml syringes
  • 25‐G needles
  • 15‐ml conical centrifuge tubes
  • BS MACS separation column (Miltenyi Biotec)
  • VarioMACS Magnet (Miltenyi Biotec)
  • Additional reagents and equipment for anesthesia of mice (unit 1.4), blood collection from mice (unit 1.7), performing a differential cell count of leukocytes (unit 14.1) and counting cells using a hemacytometer ( appendix 3A)
    Table 4.5.1   MaterialsAntibody Cocktail

    Antibody Antibody‐to‐leukocyte ratio Target cell
    CD2 1.5 µg/106 lymphocytes T + B lymphocytes
    CD5 2 µg/106 lymphocytes T (+B) lymphocytes
    CD45R 10 µg/106 lymphocytes B (+T) lymphocytes
    F4/80 2 µg/106 monocytes mature monocytes
    ICAM‐1 0.6 µg/106 leukocytes lymphocytes + monocytes

Basic Protocol 3: Chemotaxis Assay

  Materials
  • Chemoattractant (e.g., fMLP, PAF, SDF)
  • Cell suspension ( protocol 1 or protocol 22)
  • RPMI 1640 medium supplemented with 0.1% (w/v) bovine serum albumin (BSA)
  • Unsupplemented RPMI 1640 medium (Invitrogen)
  • Turk's solution (see recipe)
  • 96‐well ChemoTx Plate System (Neuro Probe, Inc., http://neuroprobe.com)
  • Cotton swabs
  • Centrifuge with microtiter plate carrier
  • Additional reagents and equipment for counting cells using a hemacytometer ( appendix 3A)

Alternate Protocol 1: Measuring Phagocyte Migration Using a Flow Chamber

  Materials
  • Murine endothelial cells (preferably primary cells, but alternatively an immortalized cell line, e.g., bEND.3) growing in 75‐cm2 culture dishes
  • Trypsin‐EDTA solution (Sigma‐Aldrich)
  • Murine recombinant TNF‐α (Sigma‐Aldrich)
  • Dulbecco's phosphate‐buffered saline (DPBS) with and without Ca2+ and Mg2+ (Invitrogen)
  • DPBS without Ca2+ and Mg2+ (Invitrogen) supplemented with 0.1% bovine serum albumin (BSA)
  • Compounds, inhibitors, or reagents of interest
  • High‐vacuum grease (Dow Corning)
  • 35‐mm circular cell culture dishes (Corning)
  • Circular flow chamber kit (GlycoTech, http://www.glycotech.com/)
  • Tygon S‐50‐HL tubing (1.6 × 4.8 × 1.6 mm; Saint‐Gobain, http://www.saint‐gobain.com/)
  • Silicone gasket (supplied with circular flow chamber kit from Glycotech; 0.025 × 0.5–cm cut‐out region)
  • Alcohol wipes
  • Diaphragm‐type vacuum pump
  • 50‐ml and 20‐ml syringes
  • Syringe pump (Harvard Apparatus)
  • Tube clamp/locking forceps
  • Inverted microscope with 20× phase‐contrast objective
  • High‐resolution CCD video camera connected to a computer
  • Graphics software (e.g., ImagePro Plus; Media Cybernetics, http://www.mediacy.com/)

Support Protocol 1: Cell Polarization Assay

  Materials
  • Nitric acid, concentrated
  • 70% ethanol
  • 50 µg/ml fibronectin (Sigma‐Aldrich) in distilled H 2O
  • RPMI 1640 medium supplemented with 0.2% (w/v) bovine serum albumin (BSA)
  • Cells to be measured in polarization assay (see Basic Protocols protocol 11 and protocol 22)
  • 1 to 10 µM fMLP (Sigma‐Aldrich)
  • 4% paraformaldehyde (see recipe)
  • Staining buffer (see recipe)
  • Primary antibody, nonconjugated
  • Phosphate‐buffered saline (PBS; Invitrogen)
  • Secondary antibody or conjugated antibodies
  • 6 µM phalloidin stock solution
  • 5 mg/ml DAPI stock solution
  • Fluorescent mounting medium
  • Coverslips
  • Beaker
  • 24‐well plate
  • Microscope slides
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Figures

Videos

Literature Cited

Literature Cited
   Alonso‐Lebrero, J.L., Serrador, J.M., Dominguez‐Jimenez, C., Barreiro, O., Luque, A., del Pozo, M.A., Snapp, K., Kansas, G., Schwartz‐Albiez, R., Furthmayr, H., Lozano, F., and Sanchez‐Madrid, F. 2000. Polarization and interaction of adhesion molecules P‐selectin glycoprotein ligand 1 and intercellular adhesion molecule 3 with moesin and ezrin in myeloid cells. Blood 95:2413‐2419.
   Bennett, W.E. and Cohn, Z.A. 1966. The isolation and selected properties of blood monocytes. J. Exp. Med. 123:145‐160.
   Boyden, S. 1962. The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes. J. Exp. Med. 115:453‐466.
   Cotter, M.J., Norman, K.E., Hellewell, P.G., and Ridger, V.C. 2001. A novel method for isolation of neutrophils from murine blood using negative immunomagnetic separation. Am. J. Pathol. 159:473‐481.
   Davies, J.Q. and Gordon, S. 2005. Isolation and culture of human macrophages. Methods Mol. Biol. 290:105‐116.
   Florey, H 1970. General Pathology. Fourth Edition. Lloyd‐Luke, London.
   Hayhoe, R.P., Kamal, A.M., Solito, E., Flower, R.J., Cooper, D., and Perretti, M. 2006. Annexin 1 and its bioactive peptide inhibit neutrophil‐endothelium interactions under flow: Indication of distinct receptor involvement. Blood 107:2123‐2130.
   Landsman, L., Varol, C., and Jung, S. 2007. Distinct differentiation potential of blood monocyte subsets in the lung. J. Immunol. 178:2000‐2007.
   Majno, G. 1977. The Healing Hand: Man and Wounds in the Ancient World. Harvard University Press, Boston.
   McGettrick, H.M., Lord, J.M., Wang, K.Q., Rainger, G.E., Buckley, C.D., and Nash, G.B. 2006. Chemokine‐ and adhesion‐dependent survival of neutrophils after transmigration through cytokine‐stimulated endothelium. J. Leukoc. Biol. 79:779‐788.
   Rocha e Silva, M. 1978. A brief survey of the history of inflammation. Agents Actions 8:45‐49.
   Taibott, J. 1968. Julius Cohnheim (1839‐1884), experimental pathologist. JAMA 206:1561‐1562.
   Yona, S., Lin, H.H., Dri, P., Davies, J.Q., Hayhoe, R.P., Lewis, S.M., Heinsbroek, S.E., Brown, K.A., Perretti, M., Hamann, J., Treacher, D.F., Gordon, S., and Stacey, M. 2007. Ligation of the adhesion‐GPCR EMR2 regulates human neutrophil function. FASEB J. 22:741‐751.
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