Array analysis software

CM generated from NB cells (CHLA-255), CAF-MSC, BM-MSC, NB/CAF-MSC and NB/BM-MSC were examined for the presence of 36 different inflammatory markers using a Human Cytokine Array (R&D System ARY005B) according to the manufacturer’s instructions. Briefly, membranes were incubated with 2 ml of CM and a cocktail of biotinylated antibodies overnight at 4°C. Following three washes, membranes were incubated in the presence of 2 ml (1:2000 dilution) of streptavidin-horseradish peroxidase (HRP) for 30 minutes at room temperature and the presence of immunocomplexes was detected by staining with 3,3′-diaminobenzidine (DAB) chromogen. Arrays were scanned and pixel density measurements were obtained using the array analysis software of LI-COR Biosciences.

NB cells were exposed to CM from NB/CAF-MSC, NB/BM-MSC or NB/Fb co-cultures for 30 minutes, washed with ice-cold PBS, harvested and lysed in RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (Thermo Scientific). The PathScan Intracellular Signaling Array Kit (Cell Signaling Technology #7744) was used according to the manufacturer’s instructions. Proteins were detected by streptavidin-conjugated DyLight^™ 680, and fluorescence images were captured using the Odyssey Infrared Imaging Systems (LI-COR Biosciences), and spot intensities were quantified using array analysis software (LI-COR Biosciences). Western blots were performed using standard protocols.