Proteome profiler human cytokine array panel a

In the primate tissue snap frozen retinal samples were either used to assess the levels of cytokine using the Proteome Profiler Human Cytokine Array Panel A (R & D Systems, Minneapolis, MN, USA) or the levels of cell stress-related proteins using the Human Cell Stress Proteome Profiler Array kit (R&D Systems, Minneapolis, MN, USA). While these arrays are for human, at both gene and protein levels there is a 93–99% homology between human and Macaque cytokines^{73 (link)}. Retinae were pooled from defined retinal regions in different primates and across whole retinae in mice. The retinae were homogenized in RIPA buffer (Millipore, UK) containing protease inhibitors (Sigma, UK). Lysates were centrifuged at 1000 × g for 10 minutes at 4 °C and the supernatant was used for the arrays and were conducted according to the manufacturer’s instructions and offer a parallel determination of 36 cytokines for the cytokine array and 26 for the human cell stress related proteins. Protein concentration was calculated using the Protein Assay (Thermo Scientific). Protein Array Analyzer for Image J was used to quantify and determine spot density. In the mouse tissues snap frozen retinae were used to assess the levels of cytokine using the Proteome Profiler Mouse Cytokine Array Panel A (R & D Systems, Minneapolis, MN, USA).

Production of a wide range of cytokines by Caco-2 cells was determined using Proteome Profiler Human Cytokine Array Panel A (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Briefly, 1 mL of pooled media from 4–7 independent experiments was mixed with 0.5 mL of Array Buffer and 15 µL of Detection Antibody Cocktail. The reaction mixture was added to previously blocked array membranes for overnight exposure. After the incubation and washing, each membrane was exposed to Streptavidin-HRP solution for 30 min and then activated by Chemi Reagent Mix. Chemiluminescence was captured on X-ray film in the form of dark dots. Afterward, the film was scanned and the optical density of dots was measured by ImageJ software (National Institutes of Health, Bethesda, MD, USA).

The collected culture media from cells stimulated with parasite and ES products were subjected to the Proteome Profiler Human Cytokine Array Panel A (R&D Systems) according to the manufacturer’s instructions. Each nitrocellulose membrane contains duplicated spots of 36 different antibodies for anticytokines, chemokines, growth factors, and adhesion proteins. Preblocked nitrocellulose membranes of the Human Cytokine Array were incubated with 1 ml of each culture media and detection antibody cocktail overnight at 4°C on a rocking platform. The membranes were washed three times with 1× Wash Buffer (R&D Systems) to remove unbound proteins. Chemiluminescent detection reagents were applied to detect spot densities. Membranes were exposed to X-ray film for 3, 5, and 10 min. Array images were analyzed using the image analysis software (Quantity One).