An agarose spot chemotaxis assay for chemokine receptor antagonists |
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Authors: | Vinader Victoria Al-Saraireh Yousef Wiggins Helen L Rappoport Joshua Z Shnyder Steve D Patterson Laurence H Afarinkia Kamyar |
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Affiliation: | aThe Institute of Cancer Therapeutics, University of Bradford, West Yorkshire BD7 1DP, UK;bSchool of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK |
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Abstract: | IntroductionChemokines are important players in directing the migration of cancer cells as part of the metastatic process. The aim of this study is to develop an easy-to-perform, reliable, and inexpensive assay for rapid analysis of anti-chemotactic activity of chemokine antagonists under a number of experimental conditions.MethodsAn agarose spot containing the chemokine chemoattractant is applied to a glass petri dish. Live cells in a media, both with and without a chemokine antagonist, are added to the dish and, following cell adhesion, the migration under the agarose spot is observed and analysed by microscopy.ResultsIn the absence of CXCL12 in the agarose, no migration under the agarose spot is detected. In the presence of CXCL12, significant migration under the agarose spot is observed which can be retarded if a neutralising monoclonal antibody or a small molecule antagonist is added to the media.DiscussionThis experimental configuration is a reliable, inexpensive and easy-to-perform chemotaxis assay, which enables assessment of the activity of CXCR4 antagonists. |
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Keywords: | Abbreviations: CXCR4, Chemokine CXC type receptor 4 CXCL12, Chemokine CXC type ligand 12 (also known as SDF1-α) FCS, foetal calf serum siRNA, small interfering ribonucleic acid mAb, monoclonal antibody GPCR, G-protein coupled receptor FPR, formylpeptide receptor |
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