Non hematopoietic panel

To measure the expression of different proteins in DCs (lower chamber) or JPCs (upper chamber) supernatants at day 1 and 7 of DC differentiation and G-CSF levels in supernatants from JPCs (cultivation in the absence of DCs) cultured with human platelet lysate (hPL) under Co/Ob conditions (with or without dexamethasone) at day 15, proteome profiler array kits (Human Cytokine Array Kit, Human Soluble Receptor Array Kit, and Non-Hematopoietic Panel; R&D Systems, Germany) were used, following the manufacturer’s instructions. Briefly, the membranes were blocked with respective array buffer for 1 h at room temperature (RT) and then incubated with 1.5 mL of sample/array buffer/detection antibody mixtures overnight at 4 °C. After washing three times, the membranes were incubated with 2 mL of diluted streptavidin–HRP at RT for 30 min. After three washing steps again, the membranes were exposed to 1 mL of the chemiluminescent reagent mixture and the radiographic films for 10 min. The developed films were scanned, and data analysis of positive signals was carried out using the ImageJ software.

To measure the expression of secreted proteins, supernatants of JPCs cultured for 15 days under untreated (CO) and osteogenic (OB) conditions, with or without dexa were analyzed using proteome profiler array kits (Human Cytokine Array Kit, Human Soluble Receptor Array Kit, and Non-Hematopoietic Panel; R&D Systems, Germany) following the manufacturer’s instructions. Briefly, the membranes were blocked with array buffer for 1 h at room temperature and then incubated with 1.5 ml of sample/array buffer/detection antibody mixtures overnight at 4°C. After washing, the membranes were incubated with 2 ml of diluted streptavidin-HRP at RT for 30 min. After three more washing steps, 1 ml of chemiluminescent reagent mixture was added to the membranes, and luminescence was detected by exposure to radiographic films (GE Healthcare, Chicago, United States) for 10 min. Developed films were scanned, and data analysis of positive signals was carried out using ImageJ software.