Human il 6 duoset

Based on a previously published protocol (Miller & Chen, 2010 (link)), whole blood from heparinized vacutainer tubes was diluted 1:10 in phosphate buffered saline and cultured with and without the endotoxin lipopolysaccharide (LPS, 50 ng/ml). A range of concentrations of hydrocortisone (0, 2.76 × 10⁻⁵ M, 2.76 × 10⁻⁶ M, 2.76 × 10⁻⁷ M, 2.76 × 10⁻⁸ M) was added to additional cultures containing LPS. Supernatants were collected and IL-6 concentration was measured in duplicate using ELISA (Human IL-6 DuoSet, R&D Systems, Minneapolis, MN). A standard range of 9.38 pg/ml to 1200 pg/ml was included on each plate and supernatants from cultures with LPS were diluted 1:10 prior to assaying while those without were assayed neat. Absorbance at wavelengths 450nm and 540 nm were measured (correction wavelength) (Molecular Devices, Sunnyvale, CA). Samples that tested above the standard range were diluted further and repeated as were samples with variance coefficients greater than 10%. Multiple measures of GRA could be constructed from the protocol, e.g., based on area under the curve, slope, inhibitory coefficient, or difference score. Analyses indicated a very high degree of overlap across these alternative measures; the GRA score used in analyses is the difference between the LPS without hydrocortisone and the LPS with the highest hydrocortisone concentration.