Quantikine elisa mouse tgf β1 immunoassay

Another portion of the right hemi-diaphragm was excised from 3-month-old and 6-month-old wt and mdx mice, immediately snap frozen in liquid nitrogen and stored at -80°C until being used to measure pro-fibrotic TGF-β1 (Transforming Growth Factor—β1) levels by enzyme linked immunosorbent assay (ELISA). The remaining diaphragm tissue was mounted and frozen for histological analysis. Briefly, diaphragm portions of 15–30 μg each, were homogenized in micro centrifuge tubes containing 200–300 μl of freshly-prepared lysis buffer (20 mM TRIS pH 8.0, 1% Triton X-100, 137 mM sodium chloride, 10% glycerol, 5 mM ethylenediaminetetraacetic acid and 1 mM phenyl methyl sulphonyl fluoride). Tissue homogenates were then centrifuged (10 min, 12000 rpm, 4°C) and supernatants were used for the ELISA (Quantikine® ELISA Mouse TGF-β1 Immunoassay, R&D Biosystems) according to manufacturer’s instructions. TGF-β1 levels were expressed as pg of TGF-β1/μg of total protein content [23 (link), 24 (link)].

Levels of active TGF-β1 in triceps were quantified using Quantikine ELISA mouse TGF-β1 immunoassay (R&D Systems, MB100B) according to the manufacturer’s recommendations and as previously described [20 (link)]. Final values were normalized to total protein concentration.