Comparative Sensitivity and Reliability of a Multi-Analytes Platform and a Multiplex Flow Cytometry in the Quantitation of Circulating Cytokines Level in a Mouse Model of Endotoxemia
Stricker-Krongrad, A., Liu, J., Bouchard, G.F., Zhong, M.
The objective of the study was to compare two different multiplex assay formats to quantify the release of the 7 circulating Th1/Th2/Th17 cytokines in plasma of lipopolysaccharide (LPS)-challenged mice.
Female C57BL6 mice (6-9 months old) were treated orally with vehicle (0.5% methyl cellulose in water) or dexamethasone (DEX, 5 mg/kg), followed by LPS (0.2 mg/kg) via intravenous injection (IV). Blood samples were collected at multiple time points after LPS challenge and were analyzed with the Rodent MAP V3.0 Antigens assay and the Mouse Cytokine Panels A & B assay (Myriad-RBM; Luminex platform), then were compared to the Mouse Th1/Th2/Th17 cytokines (Becton-Dickinson: BD CBA) assay on a BD Accuri C6 flow cytometer platform.
Per assays with Myriad-RBM Luminex platform, tumor necrosis factor α (TNF-α), interferon γ (IFN-γ), and interleukins (IL) 2, 4, 6, and 10 were shown to be the early phase responders and IL-17 to be a sustained responder. (Table 1) Pre-treatment with
DEX reduced expression of the 7 circulating Th1/Th2/Th17 cytokines. (Table 2 and 3) Plasma levels of IL-4 were detected with the Mouse Cytokine Panels A and B assay, but were below the lower limit of quantification (LLOQ) of the Rodent MAP V3.0 Antigen assay.
The 2- and 4- hour plasma samples were quantified with the BD CBA cytokine assay. IL-2 and IL-4 were below the lower limit of detection (LLOD) and IL-17A was below the LLOQ. The other 4 cytokines (TNF-α, IFN-γ, IL-6 and IL-10) were detected within the
defined concentration ranges. IL-6 was the only cytokine that should be quantitated in the diluted plasma. Variations in concentrations of cytokines were consistent and acceptable in diluted 2-hour plasma. (Table 5)
Variations were observed between the two different multiplex assay platforms in terms of cytokine concentrations, time course effects of LPS, and magnitudes of DEX inhibition. Reproducible circulating IL-6 was obtained for plasma samples of the LPStreated mice with the assays from both Myriad-RBM and BD Biosciences. The BD CBA cytokine assay was not as sensitive as the Myriad-RBM assays in detecting and quantitating circulating IL-2 and IL-10 and IL-17A levels in the LPS-treated mice, but was more reliable in measuring circulating IL-4 and TNF-α and IFN-γ levels.