Multiplexed Cytokine Profiling in Rats

A MILLIPLEX^® rat cytokine/chemokine magnetic bead panel (Sigma Millipore, Cat # RECYTMAG-65K) utilizing antibodies against IL-1β, IL-4, IL-6, IL-10, and TNF-α was used to quantify circulating inflammatory cytokines. IL-6, TNF-α, and IL-1β were selected as common pro-inflammatory cytokines [90 (link)–92 (link)], while IL-10 and IL-4 were selected as common anti-inflammatory cytokines [93 (link), 94 (link)]. An 18-hr overnight incubation was performed according to manufacturer instructions. All samples were diluted 1:2 in assay buffer prior to running the assay according to manufacturer’s instructions. Samples were run in duplicate and cytokines were measured on Luminex^® 200^™ using xPONENT^® software version 4.3 (Luminex Corporation, Austin, TX). Quality control values for each cytokine were within the range provided by the manufacturer. The ratio of IL-6/IL-10 was used to investigate the overall relationship between pro- and anti-inflammatory cytokines [95 (link)]. Due to limited plasma availability, no analyses of normoxic MT plasma were performed.