Human cytokine array panel a kit

The cytokine assay was performed using a Human Cytokine Array Panel A kit (R&D Systems) according to the manufacturer’s instructions. Briefly, serum-starved cells were treated with CLB₂ for 4 h. After the treatment, the supernatant was assayed according to the kit instructions.

USC-TA⁺, USC-TA⁻, BMSCs were cultured alone, cultured in direct contacting with PBMNCs, and with PBMNCs in 0.4 μm Millicell hanging cell culture transwell insert (Millipore, USA), respectively, to determine the cytokines and chemokines released by the cells. The levels of cytokines and chemokines were tested in cell culture supernatants using the Human Cytokine Array Panel A kit (R&D Systems Inc., MN, USA), according to the manufacturer’s instructions. The array panel template is showed in Table 2. Aliquot of 1 mL from each supernatant was incubated with 15 µL of human cytokine antibody cocktail for 1 h at room temperature, and the mixture was then added to previously blocked nitrocellulose membranes and incubated at 4 °C overnight on a rocking platform shaker. On the next day, the membranes were washed three times (10 min each) with 1× wash buffer and incubated with Streptavidin-HRP and Chemi Reagent Mix for 30 min at room temperature on a rocking platform shaker. The immunoblot images were visualized and captured using the LAS-3000 Image Analyzer (Fujifilm, Japan). Quantity One software (Bio-Rad, CA, USA, RRID:SCR_014280) was used to quantify the cytokine levels.