4 methylumbelliferone sodium salt

The medium for the experiments was prepared with 1 liter CHO medium (CD-CHO cat. # 10743–011; Invitrogen) that was supplemented with 10 ml hypoxanthine/thymidine supplement (HT 100 ×, cat. # 11067–030; Invitrogen), 40 ml L-Glutamine solution (final concentration 8 mM; L-Glutamine 200 mM; cat. # G6152-100G; Sigma), 2 grams D-(+)-glucose (cat. # G6152-100G; Sigma), 10 ml non-essential amino acids (cat. # 11140–050; Invitrogen), 10 ml MEM vitamin solution (cat. # 11120–052; Invitrogen). In addition, 4-methylumbelliferone sodium salt (M1508; Sigma) was added at 50 μM to decrease hyaluronan synthesis [38 (link)]. Also, heparin (H4784, Sigma) was added to a concentration of 250 μg/ml to promote suspension adaptation of TSG-6 stable cell lines and to increase the recovery rate of rhTSG-6 proteins [39 (link)].
Clones were each plated in two 150 mm diameter dishes at 3,000 cells/cm² in OCDPF containing 100 μg/ml of Zeocin^TM selection reagent. After 2 days, cells were washed with PBS and then were incubated in fresh OCDPF media for an additional day. The cells were washed with PBS and lifted using trypsin. After centrifugation, the cells were re-seeded at about 6 × 10⁴/ml in 1 liter of OCDPF medium for suspension culture in a spinner bottle and further cultured for 3 days.

26 × 10⁴ cells were plated in 60 mm plates. After 24 h, they were cultivated in EBSS for 4 h and collected by scraping. Cell lysis was performed in PBS plus protease inhibitors by sonication (10 s). Beta-galactosidase activity was assayed by resuspending cell lysates (5 µg) in 50 mM acetate buffer pH 4.5 plus 0.0025% v/v Triton X-100 (final volume 100 µL) and 500 µM 4-methylumbelliferyl beta-D-galactoside (Sigma-Aldrich), for 10 min at 37 °C. Beta-hexosaminidase activity was assayed resuspending cell lysates (5 µg) in 50 mM citrate buffer pH 4.5 plus 0.0025% v/v Triton X-100 (final volume 100 µL) and 500 µM 4-methylumbelliferyl-N-acetil-β-D-glucosaminide (Sigma Aldrich), for 10 min at 37 °C. Enzymatic reactions were stopped by adding 25 volumes of 0.2 M glycine pH 10.8. 4-Methylumbelliferone sodium salt (Sigma-Aldrich) was employed for calibration. Enzimatic activity was evaluated by spectrofluorimetric measurement of the 4-Methylumbelliferone released, with excitation at 365 nm and emission at 445 nm, employing NanoDrop ND-3000 (ThermoFisher Scientific). Data were expressed as nmol Methylumbelliferone/mg protein × h.