Mb100b

Blood was collected by cardiac puncture, coagulated at room temperature before centrifugation at 1500g for 20 min. Serum was collected and immediately analyzed for serum alanine transaminase [29 (link)], aspartate transaminase (AST), γ-glutamyl-transferase, bile acid, bilirubin, and albumin concentrations at The Central Laboratory, Department of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science. Serum levels of TGF-β were assessed using enzyme-linked immunosorbent assay (#MB100B, R&D systems, Minneapolis, MN, USA) according to manufacturer’s procedure.
Blood was collected from 4.5-month-old male WT and Glmp^gt/gt mice by cardiac puncture into micro-vessel EDTA tubes (Terumo Europe, Leuven, Belgium). Hematological analyses were performed by The Central Laboratory, Department of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science.

We measured the abundance of IL-6, TNF-α, TGF-β1 and sIL-6Rα in sera of mice with mouse ELISA kits of IL-6 (M6000B, R&D Systems), TNF-α (MTA00B, R&D Systems), TGF-β1 (MB100B, R&D Systems), and sIL-6Rα (DY1830, R&D Systems), respectively. The abundance of IL-6 in human sera and ascites was measured by a human ELISA kits of IL-6 (D6050, R&D Systems).

The Achilles tendon samples were cleaned of surrounding adipose tissues, rinsed with Phosphate‐Buffered Saline (PBS, 40 µL per mouse), and cut into 0.5–1 mm pieces in PBS. An equal volume of RIPA buffer (89900, Thermo Fisher Scientific) containing protease inhibitors (78442, Thermo Fisher Scientific) was added. The tendon tissues were lysed on ice for 30 min with gentle agitation followed by centrifugation to remove debris. The total protein concentration was quantified using a total protein assay (23225, Thermo Fisher Scientific). To normalize the total protein concentration, the samples were diluted to same concentration which fell within the linear range of the standard curve of the TGF‐β1 ELISA Development kits (human: R&D Systems, DB100B; mouse: R&D Systems, MB100B). The concentrations of total and active TGF‐β1 were examined immediately after dilution following the manufacturer's instructions and obtained by comparing to the standard curve and shown as mass vs volume (pg mL⁻¹). Specifically, for total TGF‐β1 measurement, samples were first treated with 1 N HCl (1 µl/50 µl supernatant) for 15 min at room temperature followed by neutralization with an equal volume of 1 N NaOH before analysis by ELISA. For the measurement of active TGF‐β1 which was the activated form of TGF‐β1 within tendons in human and mice, samples were analyzed by ELISA without acid treatment.