Flow chamber adhesion assay for measuring arrest function of chemokines
Lab/Group: Weber Lab (RWTH Aachen University), Bernhagen Lab (RTWH Aachen University)
Related Journal & Article Information
Journal: Nature Medicine
Article Title: MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment
Introduction
Chemokines recruit blood cells to sites of inflammation, where they adhere to and transmigrate through the vascular endothelium. Sustained high chemokine expression eventually leads to plaque formation. We have identified MIF as an atypical chemokine and noncognate CXC chemokine receptor ligand involved in atherosclerosis, acting through interaction the chemokine receptors CXCR2 and CXCR4 (ref. 1). Applying an in vitro adhesion assay, we have demonstrated that MIF induces monocyte arrest under shear stress.
Materials
Reagents
Primary monocytes or monocytic cell lines (e.g. MonoMac6)
Endothelial cells (e.g. human aortic endothelial cells, HAoECs)
Calcein-AM
Receptor- or chemokine-blocking antibodies
Ca2+/Mg2+
Assay buffer:
1× HBSS, 10 mmol/L HEPES, 0.5% bovine serum albumin (BSA)
Equipment
35 mm dishes
Flow chamber (proprietary item; commercially obtainable through corresponding authors)
Syringe pump
Microscope with a long integration 3CCD video camera
Procedure
1. Grow endothelial cells in 35 mm dishes until confluence. For coating dishes with adhesion molecules (if applicable), incubate dishes with PBS plus e.g. VCAM-1.Fc (0.5 µg/mL) for 4 h at 37 °C.
2. Incubate cell monolayers with the respective chemokine, e.g. 50 ng/mL MIF for 2 h or add MIF directly to the leukocytes (i.e. 2 min pre-incubation) at the beginning of the flow experiment.
3. When using monoclonal antibodies (mAb) blocking chemokines, incubate endothelial monolayers with mAbs or isotype control 30 min prior to the experiment.
4. Incubate leukocytes (1×106/mL) with calcein-AM (1-5 µM in assay buffer) for 30 min at 37 °C.
5. When using mAbs blocking the chemokine receptors, incubate cells with the respective antibody or isotype control in assay buffer for 30 min at 37 °C.
6. Wash and resuspend cells in assay buffer (0.5-1×106/mL) and keep at 37°C.
7. Assess 35 mm dish as lower wall of the flow chamber and mount on a microscope stage essentially as described by Schober et al.2 and Weber et al.3.
8. Add one volume of assay buffer containing 2 mM Ca2+/Mg2+ (final concentration: 1 mM Ca2+/Mg2+) to the leukocyte samples and perfuse at 1.5 dyne/cm2.
9. After 2-5 min, quantify the number of adherent cells in multiple fields (0.24 mm2 each, >10 per treatment) by analysis of images recorded with a long integration 3CCD video camera.
Troubleshooting
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Critical Steps
Avoid air bubbles in the chamber and tubing to guarantee a steady laminar flow.
Anticipated Results
See for example Bernhagen et al.1
References
[1] Bernhagen, J. et al. (2007) MIF is a non-cognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment. Nat. Med. Epub ahead of print.
[2] Schober, A., Bernhagen, J., Thiele, M., Zeiffer, U., Knarren, S., Roller, M., Bucala, R. and Weber, C. (2004) Stabilization of atherosclerotic plaques by blockade of macrophage migration inhibitory factor after vascular injury in apolipoprotein E-deficient mice. Circulation 109, 380-5.
[3] Weber, C., Alon, R., Moser, B. and Springer, T.A. (1996) Sequential regulation of alpha 4 beta 1 and alpha 5 beta 1 integrin avidity by CC chemokines in monocytes: implications for transendothelial chemotaxis. J. Cell Biol. 134, 1063-73.
Acknowledgements
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Keywords
leukocyte adhesion assay, chemokine, monocyte arrest