Proteome profiler mouse cytokine array panel a array kit

To measure cytokine production we used the Proteome Profiler Mouse Cytokine Array Panel A Array Kit (R&D Systems Inc.), which consists of individual nitrocellulose membranes each containing 40 capture antibodies spotted in duplicates and simultaneously profiles the relative levels of multiple cytokines in the samples. Briefly, immortalized BMDMs-WT were seeded overnight at 37°C in 5% CO₂ in 6-well plates (1×10⁶ cells/well) in a final volume of 2 ml per well. Then, cells were primed with IFN-γ (12.5 U/ml) for 2 hours, then left untreated (medium alone) or treated with nHZ, and incubated for further 24 h. Then, cell supernatants were collected, centrifuged, and then mixed with a cocktail of biotinylated detection antibodies, and subjected to the cytokine array. The streptavidin-HRP and chemiluminescent detection reagents were added sequentially, and the secreted cytokines were detected. The films were scanned (Camag Reprostar 3) and the spot area was determined using densitometric analysis software.