Chondrocytes at P1 were seeded on coverslips onside 6‐well plates (2 × 104/well) and treated by IL‐1β for 48 hours, then fixed by 4% (v/v) paraformaldehyde, stained with Safranin O (SO, Sigma) for histological evaluation of cell morphology and GAG production. Chondrocytes seeded in 6‐well plates were treated by IL‐1β for 96 hours, digested by proteinase K (Solarbio). The enzymatic hydrolysates were centrifuged. DNA content was measured by a spectrofluorometer using Hoechst 33258 dye at 460 nm (emission) and 360 nm (excitation). A series of diluted chondrocytes (1 × 106 cells) were used as a control.24 The total intracellular sGAG secretion was qualitied spectrophotometrically at 525 nm using 1,9‐dimethylmethylene blue dye (Sigma) with chondroitin sulphate (Sigma) as a standard, and normalized to the total DNA content.
1 9 dimethylmethylene blue dye
Manufactured by Merck Group
1,9‐dimethylmethylene blue dye is a synthetic dye compound used in various laboratory applications. It serves as a colorimetric indicator, detecting and measuring the presence or concentration of certain analytes. The dye has a characteristic blue color and is commonly employed in analytical techniques without further interpretation of its intended use.
Automatically generated - may contain errors
3 protocols using 1 9 dimethylmethylene blue dye
1
Chondrocyte Morphology and GAG Analysis
2
Quantifying Construct GAG Content
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Representative constructs from each replicate were homogenized overnight in a papain-containing digest buffer (Sigma-Aldrich) at 60°C. Cumulative GAG content within the construct was quantified by the 1,9 dimethylmethylene blue dye (Sigma-Aldrich) colorimetric assay,31 (link) using chondroitin sulfate (Sigma-Aldrich) as a reference standard. Data were normalized to total DNA content as determined by a Nanodrop spectrophotometer. Data are reported as μg GAG/μg DNA.
3
Chondrocyte Glycosaminoglycan and DNA Quantification
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After culture (2, 4, and 6 days), the cells removed from the old media were digested
with a proteinase K solution (Sigma) for the following biochemical assays.
Intracellular secretion of glycosaminoglycans (GAGs) was assayed by the
1,9-dimethylmethylene blue dye (Sigma) and DNA content was quantified using the
Hoechst 33258 dye (Sigma) assay. In each sample, absorbance (460 nm) of total
intracellular DNA content, as indicated by the Hoechst 33258 dye, was measured using
a spectrofluorometer. Absorbance (525 nm) of total intracellular GAG secretion was
measured spectrophotometrically and converted into concentration using a chondroitin
sulfate (Sigma) standard curve. Production of GAGs by each chondrocyte was normalized
to the total DNA content of all chondrocytes, which displayed their biosynthetic
activity in the various culture media.
with a proteinase K solution (Sigma) for the following biochemical assays.
Intracellular secretion of glycosaminoglycans (GAGs) was assayed by the
1,9-dimethylmethylene blue dye (Sigma) and DNA content was quantified using the
Hoechst 33258 dye (Sigma) assay. In each sample, absorbance (460 nm) of total
intracellular DNA content, as indicated by the Hoechst 33258 dye, was measured using
a spectrofluorometer. Absorbance (525 nm) of total intracellular GAG secretion was
measured spectrophotometrically and converted into concentration using a chondroitin
sulfate (Sigma) standard curve. Production of GAGs by each chondrocyte was normalized
to the total DNA content of all chondrocytes, which displayed their biosynthetic
activity in the various culture media.
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