Levels of TSG-6 protein in the medium collected from hASC were determined by ELISA as previously described with modifications [22 (link)]. A 96-well plate (Maxisorp; Nunc) was coated with 50 μl of 10 μg/ml TSG-6 antibody (clone A38.1.20; Santa Cruz Biotechnology) in 0.2 M sodium bicarbonate buffer (pH 9.2) overnight at 4°C. Plates were washed with washing buffer (R&D systems) after this and all subsequent steps. Nonspecific sites were blocked with 0.25% BSA in PBS/0.05% Tween (blocking buffer) for 1 hr at room temperature. Samples of 50 μl or standards of rhTSG-6 protein (R&D systems) in dilution buffer were added and incubated for 2 hrs at room temperature, followed by 50 μl/well of 0.5 μg/ml biotinylated anti-hTSG-6 antibody (R&D systems) in blocking buffer for 2 hrs at room temperature. Bound antibody was detected by incubation for 20 min with streptavidin-horseradish peroxidase (R&D systems), diluted 1:200 in PBS and then with substrate solution (R&D systems) for 20 min. Absorbance at 450 nm and 584 nm was measured by spectrophotometer (SpectraMax M5; Molecular Devices, Sunnyvale, CA, http://www.moleculardevices.com/).