Alpha-L beta-2 integrin activation on MonoMac6 cells

doi:10.1038/nprot.2007.190

Method Article

Alpha-L beta-2 integrin activation on MonoMac6 cells

https://doi.org/10.1038/nprot.2007.190

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Biological techniques

Biochemistry

Computational biology and bioinformatics

Chemical biology

Drug discovery

Cell biology

Developmental biology

integrins

chemokines

adhesion molecules

beta2-integrin

In our study, we have discovered chemokine-like functions for the cytokine MIF such as induction of leukocyte arrest on endothelial cells. Cell adhesion requires the activation, i.e. by arrest chemokines, of integrins on the cell surface, which subsequently interact with adhesion molecules, such as VCAM-1 and ICAM-1. The latter interacts with the alpha-L beta-2 integrin. As MIF, like CXCL8, induced monocyte arrest on ICAM-1-expressing CHO cells¹, we investigated the direct effect of MIF on integrin activation.

MonoMac6 cells

327C: a monoclonal antibody, which binds to the activated extended conformation of the αLβ2 integrin

Secondary antibody, e.g. FITC-conjugated

Mg²⁺ and EGTA (ethylene glycol bis(2-aminoethyl-ether)-N,N,N'N'-tetraacetic acid) as positive control

4% paraformaldehyde in phosphate-buffered saline (PFA)

Assay buffer: 1× HBSS, 10 mmol/L HEPES, 0.5% bovine serum albumin/BSA

For example using FACSCalibur (BD Biosciences, San Jose, CA)

Keep MonoMac6 cells (5×10⁵ cells/mL) on ice in assay buffer until use.
Add one volume of assay buffer containing 100 ng/mL MIF (final concentration: 50 ng/mL) or canonical chemokine plus 2 mM Ca²⁺/Mg²⁺ (final concentration: 1 mM Ca²⁺/Mg²⁺) and incubate at 37 ˚C up to 30 min.
Negative control: incubate one sample (5×10⁵ cells/mL) without stimulus for 30 min at 37 ˚C.
Positive control: incubate one sample (5×10⁵ cells/mL) with 3 mM Mg²⁺ and 1 mM EGTA
Withdraw samples at different times and add to one volume 4% PFA (final concentration: 2%)
Wash cells with PBS and incubate with the monoclonal antibody 327C (10 µg/mL) for 30 min.
Wash cells with PBS and incubate with anti-mouse-IgG FITC conjugate for another 30 min.
Measure the mean fluorescent intensity (MFI) of each sample via FACS as described by Shamri et al.². The MFI corresponds with the integrin activation.

3 h

Work fast, because integrin activation already occurs within a minute after stimulus addition.

See for example Bernhagen et al. (Nat. Med. 2007).

Bernhagen, J. et al. MIF is a non-cognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment. Nat. Med. Epub ahead of print(2007).
Shamri, R. et al. Lymphocyte arrest requires instantaneous induction of an extended LFA-1 conformation mediated by endothelium-bound chemokines. Nat. Immunol. 6, 497-506 (2005).

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Alpha-L beta-2 integrin activation on MonoMac6 cells

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Alpha-L beta-2 integrin activation on MonoMac6 cells

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Introduction

Reagents

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Timing

Critical Steps

Anticipated Results

References

Associated Publications

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Privacy Policy

Terms of Service

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