Cytokine antibody array kit

The amounts of total 42 molecules released by neurons under the GOSD condition (for 6 h) were determined by using a cytokine antibody array kit (H0128003, RayBiotech). The procedures used were based on the protocol provided by the company. The molecules selected to be analyzed were due to their functions in the regulation of cell growth, angiogenesis, or inflammation. In brief, NCM was freshly prepared by collecting 6 ml of the supernatants from GOSD-treated neuronal cultures (6.4x10⁶/dish in 10-cm dish plates) at 6 h after GOSD treatment. The reason to choose the 6 h of NCM was due to the fact at this time point, NCM has been proven to be able to protect neurons and glial cells from GOSD-induced cell injury. The numbers in Table 1 represent the content changes of the indicated factors in NCM over the same factors in the neuronal culture medium under the normoxic condition for 6 h (in fold)

Cytokine profiles in the CM of MQ, A-MQ, and O-MQ were analyzed using a cytokine antibody array kit (RayBiotech, Norcross, GA, USA) following the manufacturer’s instructions. The membranes were blocked for 30 min, then incubated for 2 h with 1 mL of CM prepared from macrophages. After washing, the membranes were incubated with biotin-conjugated antibodies for 2 h and then with horseradish peroxidase-conjugated streptavidin for an additional 2 h. The signals were viewed using a chemiluminescence detection system and analyzed using Image Quant Las-4000 (GE Healthcare Life Science, Tokyo, Japan).