Dimethylmethylene blue

Manufactured by Merck Group

Sourced in United States

Dimethylmethylene blue is a colorimetric reagent used for the detection and quantification of sulfated glycosaminoglycans (GAGs) in various biological samples. It functions by forming a stable blue-colored complex with sulfated GAGs, allowing for their spectrophotometric measurement.

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21 protocols using dimethylmethylene blue

Quantifying GAG Content in Cartilage Samples

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To quantify the content of GAG in the samples, the reaction of GAG with dimethylmethylene (DMMB) blue was measured. This technique relies on the absorption spectra of the change in 1,9-dimethylmethylene dye due to the induction of metachromasia when it binds to sulfated GAGs.⁵¹ (link)
Frozen cartilage sections were digested overnight at 56 °C in 500 μl 1 mg/ml proteinase-K solution in digestion buffer (Tris/EDTA buffer). dimethylmethylene blue chloride (16 μg/ml dimethylmethylene blue) (Sigma-Aldrich, USA) in a solution of 3.04 mg/ml glycine and 2.37 mg/ml NaCl dissolved in 0.01M HCl in d₂H₂O (pH 3) was added directly followed by the measurement of the absorbance at 520 nm using a Varioskan Flash Multimode Reader (Thermo Scientific). Chondroitin sulphate-B (Sigma-Aldrich, USA) was used to generate a standard curve, and GAG measurements were normalized to the wet weight.⁵² (link) An ANOVA with a selected significance threshold of p < 0.05 was used to determine statistically significant differences between the GAG content for each layer of cartilage.

Baer K., Kieser S., Schon B., Rajendran K., ten Harkel T., Ramyar M., Löbker C., Bateman C., Butler A., Raja A., Hooper G., Anderson N, & Woodfield T. (2021). Spectral CT imaging of human osteoarthritic cartilage via quantitative assessment of glycosaminoglycan content using multiple contrast agents. APL Bioengineering, 5(2), 026101.

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Quantifying Cartilage Proteoglycan Content

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Proteoglycan content in the cartilaginous tissue was measured by DMMB (dimethyl-methylene blue) assay, as described before [42 (link)]. For this, pellets (N = 2 per donor) were harvested at day 28 of the chondrogenic induction, washed with PBS, and digested overnight in 1 ml of lysis buffer containing 50 mM Tris-HCl (pH 8.0), and 1 mM CaCl₂ with 500 μg/ml Proteinase K (Roche, Mannheim, Germany) at 60 °C. Pellets digests (30 μl) were mixed with 200 μl of DMMB solution (38 μM dimethyl-methylene blue, Sigma, 40 mM glycine, 40 mM NaCl). Proteoglycan content was measured by spectrophotometry at 540 nm and quantified using a standard curve built with chondroitin sulphate substrate standard. The values were normalized to DNA amount in lysed cells measured using Quanti-iT PicoGreen dsDNA kit (Invitrogen, Eugene, USA). For this, 20 μl of the digested pellet sample were mixed with 80 μl TE buffer (200 mM Tris-HCl (pH 7.4), 20 mM EDTA) and PicoGreen solution, and fluorescence in samples was measured at 485/535 nm.

Melnik S., Gabler J., Dreher S.I., Hecht N., Hofmann N., Großner T, & Richter W. (2020). MiR-218 affects hypertrophic differentiation of human mesenchymal stromal cells during chondrogenesis via targeting RUNX2, MEF2C, and COL10A1. Stem Cell Research & Therapy, 11, 532.

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Quantifying SMC Proliferation and GAG Content

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Samples were digested using 125 μg/mL Papain (Spectrum, Gardena, CA) for 24 h at 60 °C. SMC proliferation was assessed, via quantification of total construct DNA, using the Quanti-iT PicoGreen DNA assay (Invitrogen, Oregon, USA). GAG content was assayed using dimethylmethylene blue (Sigma Aldrich, St Louis MO, USA).^{15 (link),16 (link),17 (link)} Acellular scaffolds were imaged under SEM as previously reported.^{14 (link)} Samples were embedded in Neg-50 media, frozen, sectioned and stained with hematoxylin and eosin using standard protocols.

Amensag S., Goldberg L., O'Malley K.A., Rush D.S., Berceli S.A, & McFetridge P.S. (2016). PILOT ASSESSMENT OF A HUMAN EXTRACELLULAR MATRIX BASED VASCULAR GRAFT IN A RABBIT MODEL. Journal of vascular surgery, 65(3), 839-847.e1.

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Quantifying GAG and DNA in Tissue Samples

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Tissue rings (N=4) and 3-ring tubes (N=3) were digested in papain solution (Sigma-Aldrich) [41 ] at 65°C. GAG and DNA contents were measured using dimethylmethylene blue (DMMB; Sigma-Aldrich) [42 (link)] and PicoGreen (Invitrogen, Carlsbad, CA) assays, respectively [43 (link)].

Dikina A.D., Strobel H.A., Lai B.P., Rolle M.W, & Alsberg E. (2015). Engineered cartilaginous tubes for tracheal tissue replacement via self-assembly and fusion of human mesenchymal stem cell constructs. Biomaterials, 52, 452-462.

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Cartilage GAG Content After Transplantation

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Tissues sampled 6 months after transplantation were subjected to a GAG assay. Osteochondral plugs within the repair area were cored out using a 4 mm internal diameter core bit fixed to a standing drill press. Cartilage slices (2 mm thick) were cut for biochemical analysis and same-thickness discs from the surrounding cartilage were harvested as controls. The samples were freeze-dried and digested in 1 ml papainase (1.25 mg/ml Papain, 100 mM Na₂HPO₄, 10 mM EDTA, and 10 mM cysteine, pH 6.3) for 18 h at 60°C. GAG content was evaluated by dimethylmethylene blue (Sigma) staining of chondroitin sulfate, as reported by Farndale et al. (1986).

Liu S., Jia Y., Yuan M., Guo W., Huang J., Zhao B., Peng J., Xu W., Lu S, & Guo Q. (2017). Repair of Osteochondral Defects Using Human Umbilical Cord Wharton's Jelly-Derived Mesenchymal Stem Cells in a Rabbit Model. BioMed Research International, 2017, 8760383.

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Nerve Tissue Composition Analysis

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Triplicate samples of each treatment were used for quantification of myelin, collagen and glycosaminoglycans (GAG) content. Each 2cm length of processed nerve was divided into 2 separate 1 cm pieces, one for histology and the other for total protein isolation.
Histology samples were fixed in 10% neutral buffered formalin and embedded in paraffin (FFPE) for histomorphometric analysis via Gomori’s Trichrome. In brief, FFPE tissues were sectioned at 5 μm, placed on slides and warmed overnight at 60°C. Slides were deparaffinized, rehydrated, and stained for Gomori’s Trichrome stain. Digital images of slides were acquired with a Nikon AZ100 M microscope at 10X magnification and collagen and myelin content was assessed using ImageJ.
Total protein was isolated by lysing nerves in protein lysis buffer (9.5 M Urea/4% 3-[(3-cholamidopropyl dimethylammonio]-1 propanesulfonate (CHAPS)/Roche Protease Inhibitor Cocktail/2.5% tributylphosphine), removing cell debris via centrifugation, and finally quantifying by Bradford Assay using Coomassie Protein Assay Reagent (Thermo Fisher Scientific, Waltham, MA) and albumin standards (Pierce, Rockford, IL). GAG content was quantified with the dimethyl methylene blue (DMMB) assay (1,9 dimethyl-methylene blue, Sigma-Aldrich, St. Louis, MO), using total protein.

Pollins A.C., Boyer R.B., Nussenbaum M, & Thayer W.P. (2018). Comparing Processed Nerve Allografts and Assessing their Capacity to Retain and Release Nerve Growth Factor. Annals of plastic surgery, 81(2), 198-202.

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DMMB Staining of FFPE Spheroid Sections

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Deparaffinize FFPE slides and rehydrate through a series of gradient ethanol to distilled water at the final. Stain the spheroid sections in DMMB staining solution [0.1% (w/v) dimethyl-methylene blue (Sigma-Aldrich) dissolved in distilled water] for 5 min followed by three times wash of distilled water. Quickly dehydrate the slides through 70 to 100% ethanol and clear in xylene. Coverslip the slides with resinous mounting medium at last.

Chan N.T., Lee M.S., Wang Y., Galipeau J., Li W.J, & Xu W. (2022). CTR9 drives osteochondral lineage differentiation of human mesenchymal stem cells via epigenetic regulation of BMP-2 signaling. Science Advances, 8(46), eadc9222.

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Quantifying Glycosaminoglycan in Chondrocytes

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The amount of GAG in cultured chondrocytes in each environment was compared using Dimethylmethylene blue (DMMB) [29 (link), 30 (link)]. The chondrocytes cultured in each environment were washed three times with PBS to remove the excess cell culture media. 0.1 M sodium phosphate monobasic (S8282, Sigma-Aldrich) and 0.1 M sodium phosphate dibasic heptahydrate were mixed to prepare 0.1 M phosphate buffer (PB), and 10 mM Na-EDTA, 10 mM L-cysteine HCl, 25wt% papain were added to prepare a papain solution. The papain solution was dispensed at 0.5 ml / well to dissolve chondrocytes, and the amount of DNA contained in each sample was quantified by The PicoGreen assay (qQuant-IT PicoGreen dsDNA Reagent, Invitrogen), and the amount of GAG was measured using a calibration curve obtained by serial dilution of shark chondroitin sulfate (C4384, Sigma-Aldrich) in Dimethylmethylene blue (DMMB, 341,088, Sigma-Aldrich) solution. The measured values were expressed as GAG amount, DNA amount, and GAG / DNA amount.

Um S.H., Seo Y., Seo H., Lee K., Park S.H., Jeon J.H., Lim J.Y., Ok M.R., Kim Y.C., Kim H., Cheon C.H., Han H.S., Edwards J.R., Kim S.W, & Jeon H. (2022). Biomimetic hydrogel blanket for conserving and recovering intrinsic cell properties. Biomaterials Research, 26, 78.

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Proteoglycan Quantification in Cartilage

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Proteoglycan content in cartilaginous tissue was measured by DMMB (dimethyl-methylene blue) assay. For this, pellets (n = 2 per donor) were harvested at day 42 of the chondrogenic induction, washed with PBS, and digested overnight in 1 ml of lysis buffer containing 50 mM Tris, pH 8.0, and 1 mM CaCl₂ with 500 μg/ml Proteinase K (Roche, Mannheim, Germany) at 60 °C. Thirty microliters of digested pellets were mixed with 200 μl of DMMB solution (38 μM dimethyl-methylene blue, Sigma, 40 mM glycine, 40 mM NaCl), and proteoglycan content was measured by spectrophotometry at 540 nm, and quantified using a standard curve built using chondroitin sulphate as a standard. The values were normalized to DNA amount in lysed cells measured with Quanti-iT PicoGreen dsDNA kit (Invitrogen, Eugene, USA). For this, 20 μl of the digested pellet sample were mixed with 80 μl TE buffer (200 mM Tris HCl, 20 mM EDTA) and PicoGreen solution, and fluorescence in samples was measured at 485/535 nm.

Melnik S., Werth N., Boeuf S., Hahn E.M., Gotterbarm T., Anton M, & Richter W. (2019). Impact of c-MYC expression on proliferation, differentiation, and risk of neoplastic transformation of human mesenchymal stromal cells. Stem Cell Research & Therapy, 10, 73.

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Quantification of GAG Content in Samples

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GAG content in culture supernatants was assessed using the Barbosa method [40 (link)]. Briefly, 250 μL of collected supernatant was incubated with 1 mL of DMMB solution (16 mg/l dimethylmethylene blue, 6 mM sodium formate, 200 mM GuHCL, all from Sigma Aldrich, pH 3.0) on a shaker at room temperature for 30 min. After centrifugation, precipitated DMMB-GAG complexes were dissolved in decomplexion solution (4 M GuHCL, 50 mM Na-Acetate, 10% Propan-1-ol, all from Sigma Aldrich, pH 6.8) at 60 °C for 15 min. Absorption was measured at 656 nm and corresponding GAG concentrations were calculated using a standard curve prepared with purified bovine chondroitin sulfate (Sigma Aldrich).
For the measurement of GAG content in cartilage tissue, samples were preliminary digested overnight at 56 °C in 1 mL of proteinase K solution (Sigma Aldrich, P2308), and 100 µL of the resulting digested solution was used for the DMMB-GAG precipitation.

Pigeot S., Bourgine P.E., Claude J., Scotti C., Papadimitropoulos A., Todorov A., Epple C., Peretti G.M, & Martin I. (2020). Orthotopic Bone Formation by Streamlined Engineering and Devitalization of Human Hypertrophic Cartilage. International Journal of Molecular Sciences, 21(19), 7233.

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