Multiplex Arrays for Quantifying Relative Levels of Cytokines in Brain Tissue Lysates: Implications for mGlu Receptor-Mediated Alterations

Abstract

Cytokines are signaling peptides important for cell–cell communication and a functional immune system. They primarily regulate inflammation in response to a trauma, providing the initial signals to recruit immune cells to the site of infection or injury as well later signals for the inflammatory response to cease and desist. In the brain, the release of cytokines can be modulated by metabotropic glutamate (mGlu) receptors. Astrocytes and microglia, the main contributors to neuroinflammation, express mGlu receptors and have been shown to fluctuate cytokine release after pharmacological mGlu manipulations. While there are multiple methods for measuring cytokine concentrations in biological samples, the assay outlined in this protocol utilizes chemiluminescence to measure relative protein levels. Captured antibodies on a nitrocellulose membrane bind the analytes from brain sample homogenates, where they are then tagged with biotinylated detection antibodies. Horseradish peroxidase and chemiluminescent reagents produce a fluorescent signal that can be imaged and quantified. The protocol outlines the process in its entirety from tissue collection to assay analysis via the protein array tool in Matlab.