Stellarray

We employed a qPCR-based gene expression system that measures the expression of 96 NF-κB dependent immunological genes (StellARray, Lonza, Basel, Switzerland). These genes are recognized for having both κB binding sites in the promoter and gene expression changes associated with increased NF-κB activity. The genes included in the array encode diverse proteins such as cytokines, chemokines, complement proteins, immunological receptors, and transcription factors (for the complete list, see Additional file
1: Table S1). We performed the qPCR array on three biological replicates for each of the following inductions (6 h treatments): untreated (vehicle), isoproterenol only (Sigma-Aldrich), TNF-α only, and isoproterenol + TNF-α. This initial exploration was performed with isoproterenol, known to be a mixed β₁ and β₂ agonist. However, the 1321 N1 cell-line expresses only the β₂-subtype
[19 (link)]. For confirmation with RT-qPCR and rat studies the selective β₂ agonist clenbuterol was preferred excluding effects mediated by other β-adrenergic subtypes.

Extraction of total RNA was performed by using RNeasy Plus Mini kit from Qiagen (Hilden, Germany) and with the optional on column DNase digestion, following the manufacturer protocol. RNA was quantified by using a Nanodrop® Spectrophotometer ND-1000 (Thermo Fisher Scientific, Wilmington, DE, USA). Synthesis of cDNA was performed by using an Omniscript Reverse Transcription Kit (Qiagen), by using 2 μg RNA and 0.1 μg/μl oligo(dT)_12-18 as primer. To investigate the apoptotic pathways, pre-coated 96-wells StellARray™ (Catalog # 00188213; Lonza Bioscience, Walkersville, MD, USA) were used and performed according to the manufacturer's protocol. Real-time PCR with 20 ng of cDNA/reaction in a final volume of 20 μl was performed in a CFX96™ Real-Time System (Bio-Rad, Solna, Sweden) by using SyberGreen Supermix (Bio-Rad). Ct-values were analysed online through Global Pattern Recognition™ analysis tool, as provided by Lonza Bioscience.