Proteome profiler cytokine array panel a

Cytokines and chemokines in microglia were detected using the Proteome Profiler Cytokine Array Panel A (R&D Systems) which recognizes cytokines, chemokines, and other soluble mediators (Table 1). The cells were cultured in 9 cm Petri dishes (11x10 6 cells) and incubated with 20 µM DHA, or control medium for 12 h and then activated with LPS (10 ng/ml) for 2.5 h. Next, the cells were lysed with M-PER Reagent according to manufacturer's instruction and lysates were incubated with the array membranes containing the assayed cytokine/chemokine antibodies for 18 h at 4°C. After several washings, the membranes were incubated with secondary antibodies conjugated with horseradish peroxidase (HRP)and exposed to HRP substrate. The images were documented by the ChemiDoc MP Imager (BioRad) and analyzed densitometrically with the ImageJ software (National Institute of Health, USA) using the Dot Blot Analyzer plugin.