Cell Biology > Adhesion

User Ratings

Your rating: None
Your rating: None
Your rating: None
 Add your comments

Monocyte and Neutrophil Isolation and Migration Assays

Simon Yona1,  Richard Hayhoe2,  Inbal Avraham‐Davidi3

1The Weizmann Institute of Science, Rehovot, Israel
2University of Cambridge, Cambridge, United Kingdom
3The 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
GO TO THE FULL TEXT:
PDF or HTML at Wiley Interscience
Are you the author of this protocol? Login or register and return to this page.

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

     
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Interscience

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: Measuring Phagocyte Migration Using a Flow Chamber
  • Support Protocol: Cell Polarization Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
  • Topics
    • Immunology
    • Cell Biology
     
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Interscience

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 14.15.1 Antibody Cocktail
    AntibodyAntibody-to-leukocyte ratioTarget cell
    CD21.5 µg/106 lymphocytesT + B lymphocytes
    CD52 µg/106 lymphocytesT (+B) lymphocytes
    CD45R10 µg/106 lymphocytesB (+T) lymphocytes
    F4/802 µg/106 monocytesmature monocytes
    ICAM-10.6 µg/106 leukocyteslymphocytes + monocytes

Basic Protocol 3: Chemotaxis Assay

 Materials
  • Chemoattractant (e.g., fMLP, PAF, SDF)
  • Cell suspension (Basic Protocol 1 or 2)
  • 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: 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- (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: Cell Polarization Assay

 Materials
  • Nitric acid, concentrated
  • 70% ethanol
  • 50 µg/ml fibronectin (Sigma-Aldrich) in distilled H2O
  • RPMI 1640 medium supplemented with 0.2% (w/v) bovine serum albumin (BSA)
  • Cells to be measured in polarization assay (see Basic Protocols 1 and 2)
  • 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
Looking for materials?  Suppliers Guide
     
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Interscience

Figures

  • Figure 14.15.1
    Purity of monocyte isolation by flow cytometry analysis. Bone marrow monocytes stained with anti-CD115 and GR-1, isolated by CD115-MACS. (A) Before isolation. (B) Following isolation.

    View Image
  • Figure 14.15.2
    Cytospin of eluted fraction illustrating neutrophil purity of cells recovered after negative selection.

    View Image
  • Figure 14.15.3
    Flow chamber assembly showing the inlet connection on the right and connections for the outlet and vacuum tubes on the left. A 35-mm diameter dish containing an endothelial cell monolayer is attached by vacuum to the base of the chamber, which is then placed on an inverted phase contrast microscope. Thus, with the gasket in place, a cell suspension can be drawn across the cut-out area from right to left and visualized through the transparent chamber.

    View Image

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.
     
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Interscience
Looking for Answers?
Do you have tips, tricks, or improvements to share?

Join the Conversation

Post new comment

The content of this field is kept private and will not be shown publicly.
CAPTCHA
This question is for testing whether you are a human visitor and to prevent automated spam submissions.