Duoset elisa

Cellular supernatants were removed and cleared by centrifugation. TRAIL and PD1 protein levels were determined by DuoSet ELISA (Biotechne, MN, USA), according to manufacturer’s instructions.

Leptin was quantified in serum using the human leptin DuoSet ELISA (DY-398, Bio-Techne, Minneapolis, MN, USA). The ELISA was carried out according to the manufacturer’s instructions using the recommended ancillary kit (Bio-Techne, DY008). Serum was diluted 1:100 in reagent diluent. Samples were run in duplicate.

Human lung epithelial A549 cells stably transfected with hIL-1R7 and hIL-1R9 encoding genes were cultured in Ham's F-12K medium containing 10% FCS. In total, 12,500 cells per well were seeded into a 384-well clear, flat-bottom, cell culture treated microplate (Corning) in 15 μl medium. After 24 h at 37 °C, 5% CO₂, medium was removed and cells were washed three times with 25 μl 1× PBS, 0.05% Tween and then resuspended in 15 μl growth medium. Antibodies were added at different concentration in a volume of 5 μl and incubated with the cells for 60 min. hIL-18 recombinant protein (MBL Co Ltd) was then added to a final concentration of 10 ng/ml in a volume of 5 μl. Cells were incubated for 6 h at 37 °C/5% CO₂. The human IL-6 cytokine concentration in the cell supernatant was determined using the DuoSet ELISA (Bio-Techne) according to the manufacturer's instructions. Fitting curves and EC₅₀ calculation were done using GraphPad Prism 8.

NE, soluble AXL (sAXL), and GAS6 levels in plasma were determined by the ELISA technique using commercial kits from R&D systems (DuoSet ELISA, Bio-techne, Minneapolis, USA) according to the manufacturer’s instructions. For determination of sAXL, the plasma form of the AXL cellular receptor, and GAS6, plasma samples were diluted 1:40 and 1:200 for NE. All samples were determined in duplicates.

CHO and 293 cells were Fugene-HD (Promega, Fitchburg, WI, USA) transfected with pcDNA.FL-TRAIL and pcDNA.sTRAIL. 48 h post-transfection, the supernatants were removed and filtered through a 0.45 μm syringe filter (Sarstedt) unless otherwise stated. Secreted TRAIL levels were determined by DuoSet ELISA (Biotechne, Minneapolis, MN, USA), according to the manufacturer’s instructions. 1 ng sTRAIL, or an equal volume of FL-TRAIL cell supernatant, was used in further assays unless otherwise stated.
For the IL-6 (Duoset, Biotechne) and CXCL5/ENA-78 ELISA (Duoset, Biotechne), PC3 cells were seeded and treated with 5 ng TRAIL or 1 ng sTRAIL unless otherwise stated. For combination treatments, the cells were pre-treated with 10 μM AKTi for 10 min and with 12.5 nM Docetaxel for 6 h before TRAIL was added for a further 72 h.
CHO cells were transfected with an empty pcDNA3 vector (ctrl), pcDNA.FL-TRAIL and pcDNA.sTRAIL. After 24 h, cells were washed and trypsinised, then counted and mixed with PC3 cells at a ratio of 1:5 for 72 h. The resulting supernatants were analysed for CXCL5/ENA-78 and IL-6 by ELISA.