Luminex xmap fluorescent detection method

Blood from all groups were collected in K₂EDTA blood collection tubes and centrifuged at 4 °C for 15 min at 2,000g within 30 min of collection. Plasma was isolated, aliquoted and stored at −80 °C until use. The EMD Millipore’s MILLIPLEX™ MAP Mouse cytokine Magnetic Bead Panel 1 kit was used for the simultaneous quantification of the following analytes: IL-1α, IL-1β, IL-2, IL-5, IL-6, IL-10, IL-13, IL-18, INF-γ and TNF-α (Merck Millipore, Darmstadt, Germany). Luminex method (12 (link)) was used. The method uses a proprietary technique to internally color code microspheres with two fluorescent dyes and to create distinctly colored bead sets of 500 5.6 µm polystyrene microspheres or 80 6.45 µm magnetic microspheres, each of which is coated with a specific capture antibody. After an analyte from a test sample is captured by the bead, a biotinylated detection antibody is introduced. The reaction mixture is then incubated with streptavidin-phycoerythrin (PE) conjugate, the reporter molecule, to complete the reaction on the surface of each microsphere. The Luminex instrument acquires and analyzes data using the LuminexxMAP fluorescent detection method and the LuminexxPONENT™ acquisition software (Thermo Fisher Scientific, Waltham, MA, USA). HMGB1 plasma levels were measured by an ELISA kit specific for rat HMGB1 (ABIN416082) according to the manufacturers' instructions.