Luminex xmap magpix

The plasma samples were isolated from the total peripheral blood by centrifugation in 500g, for 10 minutes, at 4°C. They were stored at -20°C. The plasma levels of ANXA1 were evaluated by enzyme-linked immunosorbent assay (ELISA). For IL-1β, IL-8 and TNF-α we used multiplex instrument LUMINEX xMAP MAGPIX (Millipore Corporation, Billerica, MA, USA). Technical procedures were performed according to manufacturer’s instructions.

The intact right eyes of all the studied groups were macerated in liquid nitrogen and placed in eppendorfs, which were added with 500 μL of protease (Protease Inhibitor Cocktail Set I, Cat. No. 53391, Millipore Corporation, CA, USA) and phosphatase (PhosphoSafe, Cat. No. 7,126-3-3, Novagen, Millipore Corporation, Billerica, CA, USA) inhibitor solution prepared according to the manufacturer’s instructions. The material was incubated for 20 min at 4 °C under constant stirring and centrifuged at 14,000 RPM for 10 min at 4 °C. The supernatants were then collected and immediately frozen at −80 °C. The protein concentration in the supernatant was measured using a Bradford assay (Bio-Rad, Hemel Hempstead, UK).
IL-1β, IL-6, IL-10, monocyte chemoattractant protein (MCP)-1, and TNF-α inflammatory mediators were quantified in the eye macerate supernatant and in blood plasma using the rat cytokine MILLIPLEX MAP Kit (RECYTMAG-65K; Millipore Corporation, Billerica, CA, USA) according to the manufacturer’s instructions and analyzed on the LUMINEX xMAP MAGPIX (Millipore Corporation, Billerica, CA, USA) equipment. The concentration of analytes was determined by MAGPIX xPONENT software (Millipore Corporation, Billerica, CA, USA). Results were expressed as mean ± SEM of cytokine concentrations (pg/mL).

Blood was collected from the eyes of the mice and was allowed to clot for at least 30 min before centrifugation for 10 min at 1000 × g. Then, the serum was removed and assayed on a multiplex LUMINEX xMAP MAGPIX instrument (Millipore Corporation). Antibody beads, controls, wash buffer, serum matrix, and standards were prepared for the MILLIPLEX® MAP Kit Mouse Th17 Magnetic Bead Panel (Millipore Corporation) following the manufacturer's instructions [28 (link)]. Concentrations of eight cytokines (IL-6, TNF-α, IL-1β, IL-17A, IL-10, IL-4, IFN-γ, and IL-2) were detected using the Mouse Th17 Magnetic Bead Panel according to its instructions. MAGPIX and xPONENT software were used to read the results.