The extend of ECM calcification correlates with the grade of ECM maturation. The cells were fixed with 70% ethanol (Serva Electrophoresis, Heidelberg, Germany) in aqua dest. for 20 min at 6 °C after the CCM was discarded, and the cells were rinsed with DPBS beforehand. The fixation solution was removed, and the cells were once more washed with aqua dest. before 0.5% Alizarin Red S (Waldeck GmbH & Co. KG, Muenster, Germany) in aqua dest. was added for 10 min at room temperature. The supernatant of the staining solution was removed, and the cells underwent another washing procedure with aqua dest. to ensure only the bound stains remained. For quantification, the colored calcium was dissolved by adding 10% cetylpyridiniumchloride (Sigma-Aldrich) solution containing 10 mM sodiumdihydrogenphosphate (AppliChem, Darmstadt, Germany). Optical density was measured with a microplate reader (Autobio PHOmo) at 570 nm. For the quantitative determination of bound Alizarin Red S, a standard dilution series ranging from 90 µg down to 0 µg Alizarin Red S solution was plotted.
Ethanol
Manufactured by Serva Electrophoresis
Sourced in Germany
Ethanol is a clear, colorless liquid used in electrophoresis applications. It serves as a solvent and denaturant, facilitating the separation and analysis of biomolecules such as proteins and nucleic acids. Ethanol is a core component in various electrophoresis procedures, contributing to the effective preparation and processing of samples.
Automatically generated - may contain errors
Lab products found in correlation
Cetylpyridinium chloride, by Merck Group (1 mentions)
Sodium dihydrogen phosphate, by AppliChem (1 mentions)
Ethanol, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Serva Electrophoresis (1 mentions)
Fraction 5 fatty acid free bsa, by Serva Electrophoresis (1 mentions)
Genejet genomic dna purification kit, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Agarose, by Merck Group (1 mentions)
Sodium dodecyl sulfate (sds), by Avantor (1 mentions)
Ammonium acetate, by Merck Group (1 mentions)
Ethylenediaminetetraacetic acid, by Merck Group (1 mentions)
Bromophenol blue, by Merck Group (1 mentions)
Multiplex pcr kit, by Qiagen (1 mentions)
Glycerol, by Lach-Ner (1 mentions)
Acetic acid, by Merck Group (1 mentions)
Ethidium bromide, by Serva Electrophoresis (1 mentions)
Isopropanol, by Serva Electrophoresis (1 mentions)
Proteinase k, by Serva Electrophoresis (1 mentions)
3 protocols using ethanol
1
Quantifying ECM Calcification Levels
2
Preparation and Standardization of FFA Solutions
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OA (purity >99%, Enzo Life Sciences, New York, USA) was dissolved in ethanol (purity >99.8%, Sigma) to prepare stock solutions of 150, 225, 450, and 900 mM. Stock solutions were diluted 1:300 with DMEM complexed with fraction V fatty acid-free BSA (SERVA Electrophoresis, Heidelberg, Germany) to prepare FFA concentrations of 0.5, 0.75, 1.5, and 3 mM. The molar FFA:BSA ratio was 5:1 and ethanol concentration was 0.23–0.32% w/w. The prepared FFA media were overlayed with nitrogen (Linde, Dublin, Ireland) and placed on a shaker overnight at 37°C. After sterile filtration (0.22 µm), stock solutions were stored at −20°C. Control media contained matching amounts of ethanol and BSA. For comparison of solvent effects, a 150 mM OA stock solution and control medium were prepared with DMSO (purity >99.5%, Sigma). Stock solution was diluted to generate 1.5 mM OA media. A suggested standard protocol for preparation of FFA solutions is given in the Supplementary Data .
3
Molecular Biology Reagents and Kits
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Phosphate buffered saline (PBS) were obtained from GIBCO, Invitrogen, USA. Sodium chloride (NaCl) from Centrohem, Serbia. Ethylenediaminetetraacetic acid (EDTA), acetic acid, ammonium acetate, agarose and bromophenol blue were purchased from Sigma-Aldrich, USA. Tris (hydroxymethyl) aminomethane (Tris) and Tris (hydroxymethyl) aminomethane hydrochloride (Tris HCl) from Alkaloid AD, Skopje. Sodium dodecyl sulfate (SDS) from J.T. Baker, Phillipsburg, New Jersey. Ethidium bromide, proteinase K, ethanol and isopropanol were obtained from SERVA, Germany. Glycerol from Lach-Ner, Czech Republic. Primers from Metabion, Germany. Four commercial kits: GeneJET Genomic DNA Purification Kit obtained from Thermo Fisher Scientific, USA; QIAGEN Multiplex PCR Kit from QIAGEN, Germany; BigDye™ Terminator v3.1 Cycle Sequencing Kit and BigDye XTerminator™ Purification Kit from Applied Biosystems, Invitrogen, USA. All solvents and chemicals were of analytical grade.
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