Encyclopedia of Experiments: Immunology
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Transcript
Take a gelatin-coated multi-well plate containing adhered human brain cancer cells expressing CXCR4.
Replace media with a calcium-sensitive dye. Intracellular esterases cleave the dye into a membrane-impermeable product that binds to calcium ions, exhibiting increased fluorescence.
Remove the dye, wash, and add buffer.
Place the plate in a fluorescence microplate reader with CXCR4-interacting compound and chemokine CXCL12-containing plates.
Using set automated parameters, inject increasing compound concentrations into test wells, followed by a specific CXCL12 concentration.
In control wells without the compound, CXCL12 binds to CXCR4.
This binding activates a signaling cascade, releasing calcium ions into the cytoplasm, where they bind the dye, intensifying fluorescence.
However, in test wells, interacting compounds inhibit CXCL12 binding to CXCR4, affecting calcium mobilization and causing a dose-dependent fluorescence decrease, confirming them as CXCR4 antagonists.
