Reader infinite m1000pro

For detection of calcium amounts of tissue patch material, a colorimetric calcium assay kit based on the cresolphthalein complexon method was used (cat. no. Cay-701220; Biomol, Hamburg, Germany). Shredded tissue patch material was pestled and lysed in 100 mM Tris-HCl buffer (pH 7) and centrifuged for 15 min at 4°C at 11,000 r/min. Afterwards, extinction of the supernatant was measured at 580 nm. Group sizes were as follows: wild-type and Bgn -/0 8 weeks n = 6, wild-type and Bgn -/0 16 weeks n = 7. Calcium amounts of cultured VICs were detected using a calcium assay kit (cat. no. KA1644; Abnova, Taipeh, Taiwan). Cells were washed with PBS and lysed in 100 mM Tris-HCl buffer (pH 7) containing 0.1% Triton-X-100 under rotation for 2 h at 4°C. Samples were centrifuged for 15 min at 4°C at 13,000 r/min. Extinction of supernatant was measured at 612 nm. Group sizes were n = 6 for each treatment. For measurements, a Tecan Reader Infinite M1000PRO was used.

For determination of biglycan core protein in cell culture supernatant of VIC, a sandwich enzyme immunoassay for in vitro quantitative measurement of biglycan was used (cat. no. SEJ226; Cloud-Clone, Katy, TX, USA). Supernatants were collected after 5 days of treatment and centrifuged for 20 min at 1000g and stored at -80°C until usage. The enzyme-linked immunosorbent assay (ELISA) was performed according to the manufacturer's instructions and extinction of the supernatants was measured at 450 nm. For measurements, a Tecan Reader Infinite M1000PRO was used. Group sizes were n = 4 for each treatment group. Data were normalized to the total protein amount of underlying cells using a DC™ Protein Assay (cat. no. 5000114; Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions.