Human fibronectin elisa kit

Collagen type I, fibronectin, and periostin were measured in PSCs by sandwich ELISA using a human collagen type I ELISA kit (cat# LS-F22003-1, LS Bio, Lynnwood, WA, USA), human fibronectin ELISA kit (cat# DY-1918-05, R&D Systems, Minneapolis, MN, USA), and human periostin ELISA kit (cat# DY3548B, R&D systems), respectively, following the manufacturers’ instructions. For the measurement of CTHRC1 using sandwich ELISA, pancreatic cancer cells and PSCs were seeded at 1 × 10⁶ cells in 100 mm dishes and cultured for 48 h. After cell debris was removed by centrifugation, the culture supernatant was harvested for the detection of CTHRC1. The plates were coated with 5 μg/mL of CR1P (anti-CTHRC1 polyclonal antibody) and incubated at 4 °C overnight before being incubated with the supernatant for 90 min. The plates were then treated with biotinylated 4H5 (anti-CTHRC1 monoclonal antibody) as the detection antibody at 37 °C for 90 min followed by Streptavidin-HRP (1:5000) at 37 °C for 30 min. CTHRC1 protein expression levels were detected by a microplate reader at the wavelengths of 450 nm and 650 nm.

Non-diseased, non-smoking human lung donor tissue was obtained (International Institute for the Advancement of Medicine), decellularized, and solubilized following protocols previously published by our group^{31 (link),33 (link),70 (link)}. The solubilized lung ECM (sECM) was aliquoted and stored at -80 °C until further use. The ECM was diluted for colorimetric assays to measure collagen, elastin, sulphated glycosaminoglycan, and hyaluronan content (Biocolor, United Kingdom). Fibronectin and laminin were quantified using the Human Laminin ELISA kit (Abcam, United Kingdom) and Human Fibronectin ELISA Kit (R&D Systems, Minneapolis, MN), respectively. To estimate spectral counts of specific collagen types and the composition of growth factors, the sECM was sent to and processed by RayBiotech (Peachtree Corners, GA) using the Human L3 Glass Slide Array and Human Growth Factor Array Q1. Additionally, total protein content of the sECM was quantified with a Pierce™ BCA Protein Assay Kit (Thermofisher, Waltham, MA). A total of six individual lung donors were decellularized, solubilized, and analyzed for the solubilized ECM content.