Quantification of Glycosaminoglycans in Cell Culture

Shark chondroitin sulfate (CS) (Sigma-Aldrich), calf thymus DNA (Invitrogen), bovine kidney heparan sulfate (Sigma-Aldrich), dermatan sulfate (Crescent Chemical Co., Islandia, NY) and sodium hyaluronate (Lifecore Biomedical, Chaska, MN) standards were prepared in 100 mM ammonium acetate (AA) (EMD). Media standards were prepared by serially diluting CS in one of four media formulations based on high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) containing phenol red (Hyclone, South Logan, UT): (1) “DMEM” consisting of only DMEM, (2) “HEPES” consisting of high glucose DMEM and 10 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulphonic acid buffer (HEPES) (Mediatech, Manassas, VA), (3) “FBS” consisting of high glucose DMEM and 10 % fetal bovine serum (FBS, Atlanta Biologicals, Lawrenceville, GA) and (4) “Total” consisting of high glucose DMEM, 10 mM HEPES, 50 μg/mL L-ascorbate 2-phosphate (Sigma-Aldrich), 1 % non-essential amino acids (NEAA) (Gibco, Grand Island, NY), 1 % insulin, transferrin, and selenous acid (ITS+) (BD Biosciences, Bedford, MA) and 0.4 mM L-proline (Sigma-Aldrich). Media standards were prepared in triplicate (n=3). CS standard curves within the linear range of sensitivity (0–50 μg/mL for cell and tissue assays, 0–25 μg/mL for media assays) were used to calculate apparent sGAG levels.