Human cytokine antibody array

By using human Cytokine Antibody Array (R&D, ARY005B) according to the manufacturer’s instructions, cytokines were detected in media of MDA-MB-231 transduced with sh-CTNND1 or sh-control. Membranes were blocked with the blocking buffer for 45 min, and then incubated with 1 mL of samples containing protease inhibitor cocktail overnight at 4 °C. After biotin-conjugated antibody and HRP-streptavidin incubation, chemiluminescence detection was performed.

Cells were incubated with 200 nM smoothened agonist (SAG) (Bioscience, Wiesbaden, Germany) in the absence of FCS for 24 h for activating the Hedghog (Hh) pathway. Immunofluorescence line-scan-based analysis and quantitative RT-PCR analysis were then performed [15 (link)].
For cytokine measurement, visceral and subcutaneous ASC in passage 3 were cultured for three days to a confluence of 90% and supernatants were taken. The levels of chemokines, cytokines, and growth factors in the supernatants were determined by applying a human cytokine antibody array according to the manufacturer’s instructions (R&D, Wiesbaden, Germany). The chemiluminescent membranes were developed using the ChemiDoc^TM MP System (Bio-Rad, Munich, Germany) and the signal intensity was assessed with ImageJ 1.49i software (National Institutes of Health, Bethesda, USA) by determining the pixel intensity of the detected spots. The signal value from the provided negative control was subtracted from every measured sample [13 (link)].
The 72 h supernatants of visceral or subcutaneous ASCs were also used for evaluating IL-6, IL-8, and TNFα via ELISA, as instructed by the manufacturers (PeproTech, Hamburg, Germany).