2.2.9. Glycosaminoglycans Purification and Quantification

Glycosaminoglycan from the culture medium or cell membrane was extracted using the protocol described in Viola et al. [28]. Briefly, after sample stimulation, conditioned media were collected as well as trypsin supernatants after cells harvesting (membrane GAGs). Samples were subjected to digestion with proteinase K (20 U/mL, Finnzymes, Espoo, Finland) and precipitation with ethanol (9:1 / ethanol:water).

HE/HS ∆-disaccharides were obtained digesting the pellet with a mix of heparinases I–II–III (form F. heparinum, Seikagaku, Tokyo, Japan) 0.5 U/mL each and then derivatized with AMAC (Sigma-Aldrich St. Louis, MO, USA). HE/HS ∆-disaccharides were analyzed and quantified by HPLC with respect to specific standards.

Intact HS GAGs were purified digesting the pellet with 0.1 U/mL U of Chondroitinase ABC for 5 h at 37 ◦C.

HS GAGs amount was calculated by means of the uronic acid content, using the van den Hoogen et al. method [29]. Briefly, 40 µL of the HS sample and 200 µL of concentrated sulfuric acid (80% *w*/*w*) were added in a 96-well plate. The plate was incubated for 1 h at 80 ◦C and, after cooling to room temperature, the background absorbance of samples was measured at 540 nm on a microplate reader (Tecan, Thermo Scientific, Waltham, MA, USA). Then, 40 µL of 3-hydroxybiphenol solution (100 µL of 100 mg/mL 3-hydroxybiphenol in DMSO mixed with 4.9 mL 80% (*v*/*v*) sulfuric acid) was added. After an overnight incubation, the absorbance was read again at 540 nm. D-Glucuronic acid (Sigma-Aldrich St. Louis, MO, USA) was used for a standard curve.
