Serum collection and cytokine multiplex assay

On specified days at, 1600 h, mice were humanely euthanized using CO₂ asphyxiation; this time was selected to maximize the effect of LDN and the circadian rhythm of cytokines. Mice were exsanguinated through terminal cardiac puncture and whole trunk blood collected, centrifuged at 14,000 r/min at 4°C, and stored at −80°C until assayed. Blood samples from two mice of the same treatment were pooled to order to obtain sufficient serum.

Serum samples were analyzed in duplicate using a Multiplex assay from Meso Scale Diagnostics (Rockville, MD). The V-Plex Mouse Inflammatory Panel 1 assay kit tested for measurable levels of the following analytes: IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, TNFα, IFNγ, and CXCL-1.