Human xl proteome profiler array

BEAS-2B cells were primed with 800 nmol/L BPDE for 12 hours, and THP-1 cells were starved in a reduction medium based on RPMI 1640 medium for 12 hours. The THP-1 cells were then resuspended in LHC-9 medium and added to the upper chamber, followed by cultivation with or without 12.5 μg/mL SiNPS for 48 hours. The culture medium was collected and cleared by centrifugation at 5000×g for 10 min. The release of cytokines and chemokines was analyzed using a Human XL Proteome Profiler™ Array, in conjunction with the Cytokine Array Kit (cat No: ARY022B, R&D Systems, USA). Export signal values and the average signals (pixel density) of the pairs of duplicate spots were determined and compared to corresponding signals on different arrays to determine the relative change between the controls and treated by BPDE and SiNPs.

The release of cytokines and chemokines in CPCs- and ACs-CM was analysed using the Human XL Proteome Profiler™ Array (R&D Systems, Minneapolis, MN, USA) according to the user’s manual. Briefly, membranes spotted with antibodies were incubated with the same amounts (50 µg/mL) of each CM overnight at 4 °C. The following day, detection antibody cocktail was added for 1 h at RT, before visualization using ECL. Quantitative analysis was performed on scanned (Epson perfection 1260 scanner, Seiko Epson Corporation, Nagano, Japan) X-ray films (Fujifilm GmbH, Düsseldorf, Germany) using the Protein Array Analyser plugin available for ImageJ software (US National Institutes of Health, Bethesda, MD, USA). For each membrane, average spot signal density was determined by densitometry, followed by background subtraction and normalization to the reference spots. For a correspondence between a specific molecule and its position in the membrane, a reference to the manufacturer’s datasheet is provided (Catalog # ARY022B).