The production of sGAG was assessed using the dimethylmethylene blue (DMMB) method (27 (link)). Cell suspension (20 µl) at the density of 4x106 cells/cm2 was mixed with 200 µl DMMB reagent (Sigma-Aldrich; Merck KGaA), and the absorbance at 525 nm was recorded using a FlexStation 3 MultiMode Microplate Reader (Molecular Devices, LLC). Total sGAG was normalized to total protein for cell division with the application of the BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.).
Dmmb reagent
Manufactured by Merck Group
Sourced in India, United States
DMMB reagent is a laboratory solution used for the quantitative determination of sulfated glycosaminoglycans (sGAG). It is a colorimetric assay that measures the absorbance of the complex formed between the DMMB dye and sGAG molecules. The reagent provides a simple and reliable method for the analysis of sGAG content in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Flexstation 3 multi mode microplate reader, by Molecular Devices (1 mentions)
Shark chondroitin sulfate, by Merck Group (1 mentions)
Glomax, by Promega (1 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Papain, by Merck Group (1 mentions)
Papain, by Sangon (1 mentions)
4 protocols using dmmb reagent
1
Quantifying Sulfated Glycosaminoglycans
2
Quantifying Chondrocyte GAG Synthesis via DMMB Assay
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Chondrocytes were cultured in 48 well plates at densities of 104 cells per well and exposed to PEMF in accordance to the exposure protocol mentioned. Postexposure, glycosaminoglycan (GAG) synthesis was quantified by the dimethyl methylene blue (DMMB) assay. The DMMB reagent (Sigma, India) was prepared as detailed by Panin et al.18 (link) and 200 µL was added to each well after removal of culture medium. Subsequently, absorbance values at 525 nm were noted.
3
Quantification of Chondrogenic GAG Deposition
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At the end of in vitro chondrogenic differentiation, performed as described in paragraph 4.8, 3D masses were analyzed for extracellular chondrogenic GAG deposition. The assay was performed by measuring the reaction between GAGs and 1,9-dimethylmethylene blue (DMMB) reagent (Sigma–Aldrich). Samples were digested overnight with 0.3 mg/mL papain solution in a phosphate/EDTA buffer, pH 6.5, at 65 °C. At the end of incubation, DMMB solution was added to the matrix extraction to develop a GAG-dye complex and spectrophotometrically read at 525 nm (Glomax, Promega Corporation, Madison, WI, USA). Total GAG content was extrapolated referring to a standard curve set up with shark chondroitin sulfate (Sigma-Aldrich, MS, USA). The experiment was repeated three times, and each sample was in triplicate. The final values were normalized to the number of cells used for each sample and represented as relative values ± SD compared with the first healthy donor (1H) chosen as reference control.
4
Quantifying Proteoglycan Content in Disc Samples
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NP samples were isolated from discs at each level at 6 months after surgery (n = 6). The PG content was quantified using the 1,9-dimethylmethylene blue DMMB assay [38 ,39 (link)]. Briefly, each lyophilized sample was digested with 125 μg/mL of papain (Sangon Inc., Shanghai; PRC) in sterile PBS, 5 mM of EDTA, and 5 mM of cysteine·HCl at pH 6.8 and 60°C overnight. After complete digestion, 20 μL of the papain digest was added to 200 μL of DMMB reagent (Sigma-Aldrich, St. Louis, Missouri, USA), and absorbance was detected at 520 nm. A Quant-iT™ PicoGreen® dsDNA Assay Kit (Invitrogen™) was used to determine DNA content in the discs, according to the manufacturer’s protocol (Thermo Fisher Scientific Inc., Waltham, MA; USA). The total sGAG in the disc for each group was normalized according to the tested amount of DNA, and the sGAG/DNA ratio was measured and reported.
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