Ultra sensitive kits

Cytokine, chemokine, and TNF receptor expression in spinal cord lysates from LysM^CreTnf^fl/fl and Tnf^fl/fl mice (naïve and 3 days survival) and Cx3cr1^CreERTnf^fl/fl and Tnf^fl/fl mice (naïve and 3 h survival) were measured using the mouse Proinflammatory V-Plex Plus Kit (IFNγ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, CXCL1, TNF), mouse TNFRI, TNFRII, RANTES (CCL5) (LysM^CreTnf^fl/fl and Tnf^fl/fl mice only) and MCP-1 (CCL2) (LysM^CreTnf^fl/fl and Tnf^fl/fl mice only) Ultra-Sensitive Kits (Mesoscale Discovery) on a SECTOR Imager 6000 Plate Reader (Mesoscale Discovery) according to the manufacturer’s instructions. Samples were diluted two-fold prior to measurement according to manufacturer’s instructions. Samples were measured in duplicates and data was analyzed using MSD Discovery Workbench software.

We evaluated the protein expression of two candidate genes identified from the microarray experiments, IL6 and CCL5 (RANTES), in BAL fluid of sarcoidosis and control subjects using ultrasensitive kits (MesoScale Discovery, Gaithersburg, MD) as per manufacturer’s instructions. The plates were read using the MSD Discovery workbench analyzer and software package. Two groups of sarcoidosis patients were studied: 1) subjects who also underwent microarray analysis of their BAL cells (sarcoidosis 1, N = 15); 2) a validation group of patients that were not part of the original microarray study (sarcoidosis 2, N = 10). P-values were calculated using the nonparametric, two-tailed Mann–Whitney test because the cytokine data were not normally distributed based on Shapiro-Wilk’s normality test (Prism v6, GraphPad Software, La Jolla, CA).