1 9 dimethylmethylene blue dmmb

For biochemical analyses (n = 3), specimens were digested in a pre-prepared papain solution containing 0.5 M EDTA, 0.05 M cysteine hydrochloride, and 1.0 mg·mL⁻¹ papain enzyme (Sigma, St. Louis, MO, USA) at 60 °C for 12 h. The aliquots of the sample digestion were used for the measurements of DNA and proteoglycan contents. DNA content was measured using a fluorescence assay. Sample digestion was kept at 37 °C for 20 min with 200.0 μL of Hoechst 33258 working solution at a concentration of 2.0 μg·mL⁻¹. The fluorescence was read at 360 nm for excitation and 460 nm for emission. The DNA content was normalized with a standard curve of calf thymus DNA (Sigma, St Louis, MO, USA). Total glycosaminoglycan (GAG) content was determined using a 1,9-dimethylmethylene blue (DMMB; Sigma, St. Louis, MO, USA) dye-binding assay with chondroitin-6-sulfate from shark (Sigma, St. Louis, MO, USA) as a standard. Briefly, 20.0 μL of sample was mixed with 200.0 μL of DMMB reagent, and absorbance was read at 525 nm.

Aggregates were fixed in 4% PBS-buffered paraformaldehyde and then infiltrated with increasing concentrations (10–30%) of sucrose. Following photographic documentation, aggregates were embedded in Tissue-Tek (Sakura, Zoeterwoude, The Netherlands) and cryosectioned at 10–12 μm with an HM 500 OM cryotome (Microm, Berlin, Germany). The metachromatic dye 1,9-dimethylmethylene blue (DMMB; Sigma) was used to detect and analyze the synthesized sulfated glycosaminoglycans (sGAG).

After chondrogenic differentiation, the GAG and DNA content of the hydrogels was determined to evaluate cartilage-like matrix formation. After 1 and 28 days of culture, the GelMA discs were cut in half. One half of each disc was weighed, freeze-dried and weighed again prior to digestion using 200 µL papain digestion buffer (0.2 M NaH₂PO₄ + 0.01 M EDTA, pH = 6.0, supplemented with 250 µg/mL papain (P3125, Sigma-Aldrich) and 1.57 mg/mL Cysteine HCL (C9768, Sigma-Aldrich) overnight at 60 °C.
For determination of GAG concentration, papain-digested samples were diluted in PBS-EDTA and 46 µM 1,9-Dimethyl-Methylene Blue (DMMB, Sigma-Aldrich) solution was added to the samples. Absorption was measured at 525 and 595 nm on a spectrophotometer [66 (link)] and the ratio was used to calculate the concentration with a chondroitin sulphate C (Sigma-Aldrich) standard curve.
DNA concentration was measured in the papain digests with Picogreen (Quant-iT, Thermo Fisher Scientific) according to the manufacturers’ instructions. In short, samples were diluted 1:20 in TE-buffer (0.5 M EDTA in 1 M Tris (1:5), pH 8) and 100 µL sample was mixed with 100 µL Picogreen reagent, incubated 5 min in the dark and fluorescence was measured with an excitation of 485 nm and emission of 520 nm. Known concentrations of λDNA were used as a reference.