Quantification of Cytokine Regulators

Frozen minced wound tissue samples were dissolved in RIPA buffer (Sigma) supplemented with protease-inhibitor cocktail (Roche) and the phosphatase inhibitors Na₃VO₄ (2 mM) and NaF (10 mM) in Lysing D columns (MP Biomedicals) subjected to three rounds of 20s cooled vibrational force. Protein yield was measured by Bradford assay and spectrophotometric analysis against a BSA-standard dilution. All ELISA assays were performed with DuoSet kits (R&D Systems) following manufacturer’s instructions. Western Blot analysis for IL-1RA was performed as earlier published [50 (link)]. A rabbit anti-IL-1RA IgG₁ antibody (Abcam #ab124962) which detects human and murine IL-1RA at a dilution of 1:1000 and a secondary HRP-coupled anti-rabbit IgG (H + L) antibody (Dianova) at a dilution of 1:10,000 was used. Equal loading was verified by actin. Chemiluminescence was detected after addition of TMB substrate (BD OptEIA) with a Vilber Fusion Fx7 (Vilber Lourmat).