Measuring expression profiles of inflammatory biomarkers is important in monitoring the polarization of immune responses; therefore, results should be independent of quantitation methods if they are to be accepted as validated clinical pathology biomarkers. To evaluate effects of differing quantitation methods, the seven major circulating Th1/Th2/Th17 cytokines interleukin 2 (IL-2), interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), IL-4, IL-6, IL-10 and IL-17A were quantified in plasma of lipopolysaccharide (LPS)-treated mice with two different multiplex platforms.
Female C57BL6 mice were treated orally with vehicle or dexamethasone, followed by LPS intravenously. Plasma samples were analyzed 0.5, 1, 2, 4, and 6 h post-LPS challenge with assays at Myriad-RBM and compared to assays performed on a BD Accuri C6 flow cytometer.
IL-17A response to LPS was limited but sustained, and the response for the remaining cytokines were early and transient; dexamethasone reduced expression of all 7 cytokines. TNF-α and IL-6 levels were similar across both assays, and IL-4 levels were generally very low. Plasma levels of remaining cytokines were variably lower with BD assays than Myriad-RBM assays.
The present findings demonstrate that quantitation of circulating biomarkers of inflammation can be achieved using multiplexed flow cytometry, but careful consideration must be taken for assay validation when cross-referencing with another multiplexed assay.
Leary S, Underwood W, Anthony R, Cartner S, Corey D, Grandin T, Greenacre CB, Gwaltney-Bran S, McCrackin MA, Meyer R. AVMA Guidelines for the Euthanasia of Animals, 2013 Edition. Schaumburg: American Veterinary Medical Association; 2013.
Biesmans S, Meert TF, Bouwknecht JA, Acton PD, Davoodi N, De Haes P, Kuijlaars J, Langlois X, Matthews LJ, Ver Donck L, et al. Systemic immune activation leads to neuroinflammation and sickness behavior in mice. Mediat Inflamm. 2013;2013:271359. CrossRef
Al-Harbi NO, Imam F, Al-Harbi MM, Ansari MA, Zoheir KM, Korashy HM, Sayed-Ahmed MM, Attia SM, Shabanah OA, Ahmad SF. Dexamethasone attenuates LPS-induced acute lung injury through inhibition of NF-kappaB, COX-2, and pro-inflammatory mediators. Immunol Investig. 2016;45(4):349–69. CrossRef
NC dP, Wang K, Wadhwa PD, Culhane JF, Nelson EL. Validation and comparison of luminex multiplex cytokine analysis kits with ELISA: determinations of a panel of nine cytokines in clinical sample culture supernatants. J Reprod Immunol. 2005;66(2):175–91. CrossRef
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