Il 1 beta human uncoated elisa kit

All separated plasma samples were stored at − 80 °C until analysis. All measurements were performed using the TNF alpha Human Uncoated ELISA Kit (Cat # 88–7346-88), IFN gamma Human Uncoated ELISA Kit (Cat # 88–7316-88), and IL-1 beta Human Uncoated ELISA Kit (Cat # 88–7261-88) obtained from Thermo Fisher Scientific (Waltham, MA, USA) according to the manufacturer's instructions.

Constructs were isolated and homogenized in M-PER™ Mammalian Protein Extraction Reagent (Thermo Fisher) containing 1X protease inhibitor cocktail (Sigma) for 30 minutes on ice to ensure complete cell lysis. Cell lysates were centrifuged at 12,000 rpm for 15 minutes at 4 °C to pellet insoluble debris, and supernatants were aliquoted and stored at −20 °C until further use. Protein expression was measured using commercial ELISA kits: IL-1 beta Human Uncoated ELISA Kit (Thermo Fisher, 88-7261-22), TNF alpha Human Uncoated ELISA Kit (Thermo Fisher, 88-7346-22), and MMP9 Human ELISA Kit (BMS2016-2) according to the manufacturer’s protocol.

THP-1 derived macrophage cells (10⁵/well), HDFs (10⁴/well), and 293/hTLR5 cells (3 × 10⁴/well) were seeded and grown overnight in 96-well plates (BD Biosciences). After 24 h of treatment, TNF-α production was assessed by means of the Human TNF Alpha Uncoated ELISA Kit (eBioscience, San Diego, CA, USA); IL-6 by the Human IL-6 ELISA MAX Deluxe Kit (BioLegend, San Diego, CA, USA); IL-8 by the Human IL-8 Uncoated ELISA Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). For NLRC4 activation, THP-1 macrophages were primed with LPS (InvivoGen, San Diego, CA, USA; 1 μg/mL) for 4 h, followed by transfection of FLA-AA and FLA-ST via Lipofectamine 2000 for 2 h. The culture supernatant was collected to assess the secreted amount of IL-1β with the IL-1 beta Human Uncoated ELISA Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The plates were then analyzed on a Synergy HTX multi-mode reader (Bio-Tek, Winooski, VT, USA) at respective wavelengths.

Quantitative detection of IL‐1β was achieved using the IL‐1 beta Human Uncoated ELISA Kit (Thermo Fisher) and performed according to the manufacturer's instructions. Supernatants of primary macrophages and THP‐1 cells were diluted in ELISA diluent 1:75 and 1:50, respectively. All samples were measured in technical duplicates and absorbance was measured in a microplate reader (Hidex Oy). All data were analyzed with Microsoft Excel (Microsoft, Redmond, WA, USA) and GraphPad Prism 8.0.2 (GraphPad, San Diego, CA, USA).