Cells were seeded into 75 mm plates (1 × 103 cells/plate) and incubated for 24 h before radiation treatment. Once visible colonies had formed (approximately 50 cells per colony) in the untreated control group (approximately 10–14 days post-seeding) the plates were washed twice in PBS before fixing the cells with the addition of 5 ml of 1,9-dimethyl-methylene blue zinc chloride double salt (Sigma-Aldrich, UK). After 45 min the plates were washed and allowed to air dry before colonies were counted. Analysis was performed by calculating plating efficiencies and survival fractions for control and treatment plates [8 (link)].
1 9 dimethyl methylene blue zinc chloride double salt
Manufactured by Merck Group
Sourced in United Kingdom
1,9-dimethyl-methylene blue zinc chloride double salt is a chemical compound used as a laboratory reagent. It is a dark blue crystalline solid that is soluble in water and various organic solvents. The compound is commonly used as a stain or indicator in various biological and analytical applications.
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Lab products found in correlation
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Acarbose, by Merck Group (1 mentions)
Ethylenediamine dihydrochloride, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Chondroitin sulfate sodium salt, by Merck Group (1 mentions)
4 methylumbelliferyl acetate, by Merck Group (1 mentions)
Synergy htx plate reader, by Agilent Technologies (1 mentions)
Chondroitin 6 sulfate, by Merck Group (1 mentions)
5 protocols using 1 9 dimethyl methylene blue zinc chloride double salt
1
Colony Formation Assay for Cell Survival
2
Enzymatic Activity Assays for Glycosaminoglycans
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The following reagents 4-methylumbelliferyl acetate, 4-methyllumbelliferyl-α-D galactopyranoside, N-acetil-D-galactosamine, 4-methyllumbelliferyl-β-D-glucopyranoside, taurodeoxycholate hydrate, acarbose, 4-methyllumbelliferyl-α-D-glucopyranoside, 4-methylumbelliferylβ-D-N,N’,N’’triacetylchitotrioside, ethylenediamine dihydrochloride, formic acid, chondroitin sulfate sodium salt from shark cartilage, 1,9-dimethyl-methylene blue zinc chloride double salt, sodium hydroxide (NaOH) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 4-methyllumbelliferyl sulfate potassium and 4-methyllumbelliferyl α-L-iduronide were purchased from Glycosynth (Warrington, UK). Sodium acetate, sodium citrate, sodium phosphate, and aminoacetic acid (glycine) were purchased from Synth (São Paulo, Brazil).
3
Quantifying Chondrogenic Differentiation via GAG
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To study the chondrogenic differentiation of the 3D pellets, the concentration of glycosaminoglycans (GAG) in the supernatant was measured at day 21 of cell culture, with the last exchange of medium being carried out 4 days before the end of the culture. Therefore, a stock solution of dimethylmethylene blue (DMMB) was prepared, consisting of 0.0021% 1.9‐dimethyl‐methylene blue zinc chloride double salt (#341088, Sigma‐ Aldrich), 0.2% sodium formate (#71541, Fluka), absolute ethanol, and distilled water. By adding formic acid, the pH was adjusted to 1.5. The DMMB was then added to the supernatant of the respective samples and within 5 minutes, the absorbance could be recorded at a wavelength of 595 nm (SpectraMax M5).
4
Quantifying Chondroitin-6-Sulfate Content
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A 500 μg/mL standard of chondroitin-6-sulfate (Sigma, St. Louis, MO) was prepared in PBE buffer (0.1 M Na2HPO4, 0.01 M Na2EDTA, pH 6.5). The 500 μg/mL standard was then serially diluted in PBE buffer to generate subsequent standards for curve fitting. Fifty microliters of each standard and papain-digested sample was pipetted into a 96 well plate in triplicate. Dimethylmethylene blue (DMMB) solution was prepared by dissolving 1 mg of 1,9-dimethylmethylene blue zinc chloride double salt (Sigma, St. Louis, MO) in 0.313 mL of 100% ethanol with 0.148 g NaCl and 0.19 g glycine added and then diluted up to 50 mL in Type I water. Two hundred fifty microliters of DMMB solution was added to each standard or sample well. Standard and sample absorbance at 525 nm was measured using a Synergy HTX plate reader (BioTek, Winooski, VT). Sample glycosaminoglycan content was calculated using the equation derived from the quadratic regression of standards’ absorbance values.
5
Radiation-Induced Colony Formation Assay
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Cells were irradiated with varying doses 24 h after seeding into 75 mm plates (1,000 cells/plate). 1,9-dimethyl-methylene blue zinc chloride double salt (Sigma-Aldrich, UK) was used to fix and stain the cells between 10 and 14 days after seeding, when colonies consisting of at least 50 cells/colony had formed in the untreated control group. The plating efficiencies and survival fractions for treatment and control colony formation (CF) groups were calculated (40 (link)).
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