Magpix xponent software

The values concerning the quantification of mast cells and neutrophils of the in vivo tissue samples were expressed as the mean ± SEM of the number of cells per mm² in three sections of 0.5 µm (leaving a space of 40 µm between each section) for each patient (n = 20 patients).
Hep-2 cells were counted using the Countess Automated Cell Counter (Invitrogen). The concentration of the cytokines was determined using MAGPIX Xponent software (Millipore Corporation). The in vitro analyses were repeated a minimum of three times.
The ultrastructural and immunocytochemistry expression of ANXA1 and FPR2 was determined using ten cells for each group investigated. The area of each cell compartment was determined using Axiovision imaging software (Zeiss). In the nucleus and cytoplasm, the density of colloidal gold particles was calculated as the mean ± SEM of the number of particles per µm². In the plasma membrane, the density of colloidal gold particles was calculated as the mean ± SEM of the number of particles per µm.
The significant differences between the means were determined using analysis of variance. This test was followed by the Bonferroni post-hoc test on select experimental groups using Graph-Pad Prism 4.0 (Graph-Pad, San Diego, CA, USA). A probability value less than 0.05 was considered to be statistically significant.

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).

Hippocampal samples were sonicated in a 50 mM Tris-HCl, 150 mM NaCl and 1% Triton-X pH 7.4 buffer containing a complete protease inhibitor cocktail and PhosSTOP tablets (Roche Applied Science, Mannheim, Germany, Cat No. 04906837001). Subsequently, samples were centrifuged at 10,000 × g for 20 min at 4 °C to obtain organ homogenates. For multiplex analysis, 25 μl of the hippocampal homogenates were employed using the MILLIPLEX MAP rat cytokine/chemokine panel (MILLIPLEX MAP RECYTMAG-65 K, Millipore Corporation, EUA, Cat No. #RECYMAG65K27PMX) and MAGPIX® Multiplexing Instrument (Millipore) according to the manufacturer’s instructions. Five analytes were studied in this work: IL-1β, IL-6, TNF-α (tumour necrosis factor-α), GRO/KC (growth-regulated alpha protein; also known as CXCL1) and MCP-1 (monocyte chemoattractant protein-1). The concentration of analytes was determined by MAGPIX Xponent software (Millipore Corporation, Billerica, MA, USA), and the results are reported as the mean ± SEM.