Cytokine elisa

Elicited peritoneal macrophages were primed with 200 ng/ml LPS for 2 h. Macrophages were then treated with or without 5 mM ATP (Sigma-Aldrich, no. A6419) for 1 h, 25 µg/ml ox-LDL for 24 h, or 200 μM palmitate-BSA for 24 h. In some experiments, after LPS priming, macrophages were treated with or without 5 mM ATP plus 3-methyladenine (3-MA; Sigma-Aldrich, no. M9281) for 1 h or 200 μM palmitate-BSA plus 3-MA for 24 h. The culture supernatants were collected and stored at −80°C for cytokine ELISA (BD Bioscience). Resident peritoneal macrophages from diet-fed Ldlr^−/− mice receiving bone marrow from WT and atg5 MSKO mice were stimulated with or without 100 ng/ml LPS for 3 h. Total RNA was isolated from macrophages using TRIzol reagent (Fisher Scientific, no. 15596026). cDNA preparation and real-time PCR were conducted as described previously (28 (link)). Total cell protein was harvested from macrophages using RIPA buffer containing protease inhibitor cocktails (Roche, no. 05892791001).

Ferrous chloride tetrahydrate (FeCl₂·4H₂O), ferric chloride hexahydrate (FeCl₃·6H₂O), aqueous ammonia (NH_3(aq)), citric acid, 3-mercaptopropionic acid (3 MPA), tellurium at 200 mesh (Te), sodium (Na), naphthalene (C₁₀H₈), and cetyltrimethylammonium bromide (CTAB) were employed as supplied from Sigma/Aldrich, SP, Brazil (new bottles, kept closed in desiccators, analytical grade). Cadmium acetate (Cd(OAc)), sodium hydroxide (NaOH), tetrahydrofuran (THF), and acetone (C₃H₆O) were employed as supplied from Labsynth, SP, Brazil. All cell culture reagents (cell media, supplements, Trypsin-EDTA) were acquired from ThermoFisher Scientific, SP, Brazil. All plasticware for cell culture was acquired from Corning, SP, Brazil. Mouse recombinant GM-CSF was acquired from R&D Systems, SP, Brazil, while Ultrapure LPS (from E. coli 0111:B4) and Nigericin were acquired from Invivogen, SP, Brazil. 4′, 6-diamidino-2-phenylindole (DAPI), glycerol-p-phenylenediamine, and all remaining salts and solutions were acquired from Sigma-Aldrich, SP, Brazil. APC-conjugated anti-mouse CD11c and fluorescein-5-isothiocyanate (FITC)-conjugated anti-mouse MHC Class II I-Ab antibodies were acquired from ThermoFisher Scientific, SP, Brazil. Cytokine ELISA were from BD Biosciences, SP, Brazil.