Cytokine Analysis of Cardiac Tissue

Cardiac tissue was collected at 14 dpi and kept frozen at −80°C until analysis. Cardiac fragments were thawed and disrupted by sonication in ice-cold extraction buffer (PBS supplemented with 1% protease cocktail inhibitor) (Roche, Basel, Switzerland) and 1% Nonidet P40 (Sigma-Aldrich). Afterward, all samples were centrifuged at 500 × g, and the supernatants were collected and used for cytokine analysis (69 (link)). Monocyte chemoattractant protein-1 (MCP-1), interferon-γ (IFN-γ), tumor necrosis factor (TNF), and interleukin-6 (IL-6) were measured using the BD cytometric bead array (CBA) mouse inflammation kit (Becton, Dickinson, New Jersey, USA) according to the manufacturer’s recommendations using a FACSCalibur device (Becton, Dickinson). Data analysis was performed using FCAP software (Becton, Dickinson). Protein concentration was determined using the Pierce BCA protein assay kit (Thermo Fisher, Waltham, MA, USA).