Horse serum

The schematic configuration of the bio-immunoassay for the quantitative measurement of ADA in serum is described in Figure 3B and was carried out as follows: ELISA plates that were coated overnight at 4°C with 5 μg/ml produced IFX-F(ab')₂ in PBS (pH 7.4). ELISA plates were then washed three times with PBST and blocked with 300 μl of 2% w/v BSA in PBS for 1 h at 37°C. Next, triplicates of 1:400 diluted serum samples were added at triplicates and serially diluted 2-fold in 2% w/v BSA in PBS, 10% horse serum (Biological Industries) and 1% Tween 20 in PBS (1:400– 1:51,200 serum dilution factor). Plates were incubated for 1 h at RT. On the same plate, serial dilutions of 10 nM ADA standard were incubated in triplicate and serially diluted 2-fold in 2% w/v BSA in PBS, 10% horse serum (Biological Industries) and 1% Tween 20 in PBS, to allow the conversion of the tested serum to units per milliliter. ELISA plates were washed three times with PBST and 50 μl of HRP conjugated anti-human IgG Fc was added to each well (50 μl, 1:5,000 ratio in 2% w/v BSA in PBS) and incubated for 1 h at RT. ELISA plate was then washed three times with PBST and developed by adding 50 μl of TMB followed by quenching with 50 μl 0.1 M sulfuric acid. Plates were read using the Epoch Microplate Spectrophotometer ELISA plate reader.

C2C12 mouse myoblasts (ATCC) were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) containing 4.5 g/L D‐glucose, 10% foetal bovine serum (Gibco) and 1% penicillin‐streptomycin solution (HyClone) in appropriate conditions. When the cells reached 90%‐100% confluence, they were incubated in DMEM containing 2% horse serum (HS, Bioind, Israel) to promote differentiation into myotubes for 5 days. The myotubes were treated with 100 ng/mL FGF19 (R&D) and 0.5 mM PA (Sigma‐Aldrich) for 24 hours. The dissolution method and concentration selection of FGF19 and PA were consistent with our previous study.²²

C2C12 myoblast and 293T cells were purchased from American Type Culture Collection (ATCC). The growth medium was Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, California, USA) containing 10% fetal bovine serum (FBS, Gibco, California, USA) and 1% penicillin–streptomycin (PS, Thermo Scientific, Massachusetts, USA), and the differentiation medium was DMED medium containing 2% horse serum (Bioind, Shanghai, China) and 1% PS. Then, the cells were cultured in a 37 °C cell incubator with 5% oxygen and 95% carbon dioxide. The transfection reagent was Lipofectamine™ 3000 (Thermo Fisher Scientific, Massachusetts, USA), operated according to its instructions.