ChemoTactics' Protocols
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Activation of chemokine receptors by their cognate ligands leads to internalization of the receptor-chemokine complexes, which can be monitored by the use of fluorescent labeled ligands. This process can be exploited to detect the receptor expression and has been successfully used to identify the cells expressing the receptors of interest within a complex mixture of primary cells.
In this protocol, biotinylated CXCL12 is pre-mixed with phycoerythrin (PE)-conjugated streptavidin and subsequently incubated with U937 cells at 37°C in the presence or absence of AMD3100, a small-molecule inhibitor of its receptor, CXCR4. CXCL12 internalization is detected by flow cytometry, and its inhibition by AMD3100 suggests the receptor-mediated endocytosis. |
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Biotinylated chemokines have a wide range of assay applications. Enzyme-Linked ImmunoSorbent Assay (ELISA) is a powerful technique that can measure the presence of a substance or chemokine with an antibody. Using different streptavidin conjugates, one can study chemokine internalization, receptor binding, or receptor expression levels. Their ability to recognize functional forms of receptors has been exploited by Abilita Bio, our collaborator, to demonstrate the correct folding of their recombinant stabilized receptor (EMP, or Enabled Membrane Protein) in an ELISA assay.
In this protocol, biotinylated CCL19 is used to measure the binding of a purified sample of stabilized CCR7 (CCR7-EMP) generated by Abilita Bio. By using streptavidin-HRP, one can construct the binding curve of CCL19 to CCR7-EMP and calculate the dissociation constant. 
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Introduction
To test the migration ability of cells in response to the simulation by chemokine. The EC50 of each chemokine will depend on the cell line and receptor. For example, to test the migration of CXCR4 expressed in Jurkat cells in response to SDF-1a, use 0.003, 0.012, 0.048, 0.019, 0.75. 3.0 nM SDF-1a. Protocol
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