Human cytokine array q4000

Cytokine analysis was performed using the Human Cytokine Array Q4000 (RayBiotech, Norcross, GA, USA) which included 200 human inflammation factors, growth factors, chemokines, cytokines and receptors. Then, a customised antibody array which included 24 prioritised proteins from the Q4000 array was analysed. Briefly, the arrays were blocked with the blocking buffer (provided by the kit) for 30 min and incubated with 1 mL of conditioned medium for 2 h at room temperature. After washing, the arrays were incubated with biotinylated antibody cocktail at room temperature for 2 h, and then with Cy3 equivalent dye‐streptavidin for another 1 h at room temperature. Detection was performed according to the manufacturer's instructions.

The concentrations of 200 serum proteins were measured simultaneously using the Human Cytokine Array Q4000 (QAH-CAA-4000, RayBiotech, Georgia, USA) as previously described (11 (link)). Briefly, after incubated with blocking buffer for 60 min at room temperature (RT), the arrays were added with 100 µL of 2-fold diluted serum and were incubated at 4°C overnight. On day 2, the arrays were added biotin-labeled detection antibody for 1 hour (h) at RT followed by extensive washing. Alexa Fluor 555-conjugated streptavidin was added and incubated for 1 h at RT. The signals were scanned, and data were extracted by an InnoScan 300 scanner (Innopsys, Carbonne, France).