Quantification of Proteoglycans and DNA in Cell Pellets

BM-MSC and SM-MSC pellets were collected and stored at -20 °C in medium prior to biochemical assays. The dimethylmethylene blue (DMMB) spectrophotometric assay (Sigma-Aldrich, St. Louis, MO) was used to quantify proteoglycan content in pellets digested in 0.5 mg/mL papain (Sigma Aldrich St. Louis, MO). Chondroitin-4 sulfate (Sigma-Aldrich, St. Louis, MO) was used to establish a standard curve and the optical density determined at 525 nm [47 (link)]. Total DNA content was determined using 0.5 mg/mL papain digested pellets incubated for 24 h at 65 °C. Digested samples were then mixed bisbenzimide compound (Hoescht, Sigma-Aldrich, Burlington, MA) and DNA was quantified using a fluorometric assay with an excitation wavelength of 348 nm and an emission wavelength of 456 nm. Calf thymus DNA (Sigma-Aldrich, St. Louis, MO) was used to establish a standard curve. Proteoglycan concentration was normalized to the quantity of DNA in that sample.