Lipopolysaccharide-induced increases in cytokines in discrete mouse brain regions are detectable using Luminex xMAP ® technology

Abstract

Methods to determine cytokine protein content in samples of interest, such as enzyme-linked immunosorbent assay (ELISA), are often labor-intensive and costly. Furthermore, because ELISA requires relatively large sample volumes and protein concentrations, it is difficult using this technique to determine protein content for multiple cytokines from individual samples. Recently, Luminex has developed an open source hardware platform combining flow cytometry- and bead-based antibody capture that is capable of detecting multiple analytes from a single sample. In the present study we employed the Luminex 200 ® platform to determine the cytokine protein content in discrete brain regions of C57BL/6J mice. In spike-and-recovery experiments, known concentrations of murine recombinant interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)α were added either singly or as a mixture of all three to whole brain homogenates containing known quantities of total protein. Spiked samples were assayed for either a single cytokine or for multiple cytokines using 1-plex or 3-plex assay kits, respectively. In whole mouse brain homogenate we recovered between 81% and 103% of the recombinant cytokines. We then injected C57BL/6J mice intraperitoneally with bacterial lipopolysaccharide (LPS) and sacrificed them 4 h later. We detected in samples taken from LPS-stimulated mice 4- to 870-fold increases in serum or spleen cytokine protein, and 1.5- to 16-fold increases in cytokine protein in discrete brain regions, relative to protein content in samples obtained from vehicle-treated animals. These results indicate that multiple cytokines may be reliably assayed from discrete regions of mouse brain using a single sample.