Mouse il 1 alpha il 1f1 duoset elisa

The analysis of cytokine expression in tumors of the treatment groups at the protein level was done using different ELISA kits. Mouse Th1/Th2 Uncoated ELISA (Invitrogen) was used to determine the protein levels of IFN-γ, IL-2 and IL-4. IL-1α, IL-12, TGF-β, PDGF-BB, VEGF and COX-2 were measured with the Mouse IL-1 alpha/IL-1F1 DuoSet ELISA, Mouse IL-12 p70 DuoSet ELISA, Mouse TGF-β1 DuoSet ELISA, Mouse/Rat PDGF-BB DuoSet ELISA, Mouse VEGF DuoSet ELISA and Human/Mouse Total COX-2 DuoSet IC ELISA, respectively (all from R&D Systems). TARC was determined with the Mouse CCL17/TARC Quantikine ELISA Kit (R&D Systems). For protein extraction, various sections (of 50 μm thickness) from each tumor were collected and protein lysates were prepared using RIPA buffer (20 mM Tris–HCl (pH 7.2), 150 mM NaCl, 2% (w/v) NP-40, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate) and cOmplete™ Protease Inhibitor Cocktail (Sigma-Aldrich) [28] (link). The total protein concentration of each tumor sample was determined using the DC protein assay (Bio-Rad Laboratories GmbH). Equal amounts of total protein (50 μg) were diluted with ELISA-Buffer (ELISA/ELISPOT Diluent, Invitrogen), or Reagent Diluent Concentrate 2 (R&D Systems), respectively. Cytokine amounts were determined according to the manufacturers' instructions.

The levels of inflammatory cytokines, including interleukin‐6 (IL‐6), IL‐1α, IL‐1β, tumor necrosis factor‐α (TNF‐α), and transforming growth factor β (TGF‐β) in the plasma and bronchoalveolar lavage fluid (BALF) were determined using respective ELISA kits—Mouse IL‐6 DuoSet ELISA (DY406; R&D), Mouse IL‐1 alpha/IL‐1F1 DuoSet ELISA (DY400; R&D), Mouse IL‐1 beta/IL‐1F2 DuoSet ELISA (DY401; R&D), Mouse TNF‐alpha DuoSet ELISA (DY410; R&D), and Mouse TGF‐beta 1 DuoSet ELISA (DY1679; R&D), as per the manufacturer's protocols.