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Lipopolysaccharide-induced increases in cytokines in discrete mouse brain regions are detectable using Luminex xMAP
1Department of Anesthesiology, University of Michigan, Ann Arbor, MI 48109, USA.
Insights
This study introduces the Luminex 200 platform for efficiently measuring multiple cytokines in mouse brain samples. This method overcomes limitations of traditional enzyme-linked immunosorbent assay (ELISA), enabling reliable analysis from single samples.
Area of Science:
- Neuroscience
- Immunology
- Biotechnology
Background:
- Traditional methods like ELISA for cytokine detection are labor-intensive, costly, and require large sample volumes.
- Detecting multiple cytokines from individual samples using ELISA is challenging due to these limitations.
Purpose of the Study:
- To evaluate the Luminex 200 platform for multiplex cytokine analysis in discrete mouse brain regions.
- To assess the reliability and efficiency of the Luminex platform compared to conventional methods.
Main Methods:
- Utilized the Luminex 200 platform for bead-based antibody capture and flow cytometry analysis.
- Performed spike-and-recovery experiments with known concentrations of murine recombinant interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)alpha.
- Assayed cytokine content in brain homogenates and serum from lipopolysaccharide (LPS)-stimulated and vehicle-treated C57BL/6J mice.
Main Results:
- Recovered 81% to 103% of spiked recombinant cytokines in whole mouse brain homogenate.
- Observed significant increases (1.5- to 16-fold) in cytokine protein within discrete brain regions of LPS-stimulated mice.
- Detected 4- to 870-fold increases in serum or spleen cytokine protein post-LPS stimulation.
Conclusions:
- The Luminex 200 platform reliably quantifies multiple cytokines from discrete mouse brain regions using single samples.
- This method offers a more efficient and sensitive alternative to ELISA for cytokine profiling in neuroscience research.
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)-1beta, IL-6, and tumor necrosis factor (TNF)alpha 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 4h 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.
