Cells were cultured in a 10-cm dish in high glucose DMEM supplemented
with 10% FBS and penicillin (100 U/ml) and streptomycin (100 μg/ml).
After washing with PBS-EB, the cells were lysed with 1 ml 0.1 M NaOH. Following,
8 μl acetic acid was added. The cell lysate was added with Pronase (2
mg/ml, Sigma-Aldrich) and digested overnight at 37°C. After inactivation
at 95°C for 10 min, the released glycosaminoglycan was enriched by
passing through a Q-column, desalting and then digested with heparin lyases I,
II, and III (Sigma-Aldrich) overnight at 37 °C. The resultant HS
disaccharides were collected by filtering the digested glycosaminoglycan though
a 3000 MW cut-off filter (Thermo-Fisher), labeled with 2-aminobenzide and then
subjected to separation by an Agilent 1260 HPLC system with a Propac PA1 (4
× 250 mm, DIONEX) column connected with a Propac PA1 Guard column (4
× 50 mm, DIONEX). The separated disaccharides were detected using a
fluorescence detector with excitation at 348 nm and emission at 440 nm.
with 10% FBS and penicillin (100 U/ml) and streptomycin (100 μg/ml).
After washing with PBS-EB, the cells were lysed with 1 ml 0.1 M NaOH. Following,
8 μl acetic acid was added. The cell lysate was added with Pronase (2
mg/ml, Sigma-Aldrich) and digested overnight at 37°C. After inactivation
at 95°C for 10 min, the released glycosaminoglycan was enriched by
passing through a Q-column, desalting and then digested with heparin lyases I,
II, and III (Sigma-Aldrich) overnight at 37 °C. The resultant HS
disaccharides were collected by filtering the digested glycosaminoglycan though
a 3000 MW cut-off filter (Thermo-Fisher), labeled with 2-aminobenzide and then
subjected to separation by an Agilent 1260 HPLC system with a Propac PA1 (4
× 250 mm, DIONEX) column connected with a Propac PA1 Guard column (4
× 50 mm, DIONEX). The separated disaccharides were detected using a
fluorescence detector with excitation at 348 nm and emission at 440 nm.