Collagen content assay was based on the quantitative dye-binding Sircol method (Biocolor, Ireland). Skin biopsies were suspended in 2 ml of a 0.5 M acetic acid—pepsin (2.5 mg/ml) solution and dissociated using UltraTurrax (vWR, France). Collagen extraction was performed overnight at 4°C under stirring. Suspension was then centrifuged at 12,000 g for 10 min and 20 μl of each sample were added to 1 ml of Syrius red reagent. Tubes were rocked at room temperature for 30 min and centrifuged at 12,000 g for 10 min. The supernatants were discarded and tubes washed with 750 μl of ice-cold salt acid. After another 12,000 g centrifugation for 10 min, the collagen-dye pellets were suspended in 1 ml of 0.5 M NaOH. Optical Density (OD) was read at 555 nm on a microplate reader (Varioskan Flash, Thermo scientific) vs. a standard range of bovine collagen type I concentrations (supplied as a sterile solution in 0.5 M acetic acid). Results were expressed as collagen content in μg/mm2 of skin.
Ultraturrax
Manufactured by Avantor
Sourced in France
The UltraTurrax is a high-speed homogenizer designed for dispersing and emulsifying samples in the laboratory. It is capable of effectively reducing the particle size of solids and breaking down agglomerates in liquids.
Automatically generated - may contain errors
4 protocols using ultraturrax
1
Quantitative Collagen Content Assay
2
Quantitative Collagen Assay in Skin
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Collagen content assay was based on the quantitative dye-binding Sircol method (Biocolor, Ireland). Skin biopsies taken from the site of injection were suspended in 2 mL of a 0.5 M acetic acid—pepsin (2.5 mg/mL) solution and dissociated using UltraTurrax (vWR, France). Collagen extraction was performed overnight at 4°C under stirring. The solution was then centrifuged at 12,000 g for 10 min and 20 μL of each sample were added to 1 mL of Syrius red reagent. Tubes were rocked at room temperature for 30 min and centrifuged at 12,000 g for 10 min. The supernatants were discarded and the tubes washed with 750 μL of ice-cold salt acid wash. After another 12,000 g centrifugation of 10 min, the collagen-dye pellets were resuspended in 1 ml of 0.5 M NaOH Alkali solution. Optical density (OD) was then read at 555 nm on a microplate reader (Varioskan Flash, Thermo scientific) vs. a standard range of bovine collagen type I concentrations (supplied as a sterile solution in 0.5 M acetic acid). Results were expressed as the collagen content in μg/mm2 of skin.
3
Xenograft Tumor Metabolomics Protocol
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For each xenograft, three different pieces (frozen tumor tissue samples,
20–50 mg) of the same tumor were taken from three different animals
(n = 3) and homogenized using an Ultra Turrax (VWR,
Pennsylvania, USA) with 10 µl/mg of extraction solvent (85:15
MeOH/H2O). The homogenized sample were stored at −80°C for
20 min and subsequently centrifuged for 15 min at 13,000g.
Supernatants were collected and used for targeted and untargeted
metabolomics analysis.
20–50 mg) of the same tumor were taken from three different animals
(n = 3) and homogenized using an Ultra Turrax (VWR,
Pennsylvania, USA) with 10 µl/mg of extraction solvent (85:15
MeOH/H2O). The homogenized sample were stored at −80°C for
20 min and subsequently centrifuged for 15 min at 13,000g.
Supernatants were collected and used for targeted and untargeted
metabolomics analysis.
4
Metabolite Extraction and Analysis of Zymoseptoria tritici
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In this study, we used three Z. tritici isolates that were previously described (26 (link)). Previous studies using axenic propagation of Z. tritici have used fungal cells propagated on yeast-malt-sucrose (YMS) medium (21 (link)), and similar growth conditions were used in this study. YMS-agar medium was prepared with 0.4% (wt/vol) yeast extract (Bacto yeast extract; Thermofisher), 0.4% (wt/vol) malt extract, and 0.4% (wt/vol) sucrose supplemented with 2% (wt/vol) Bacto agar. The fungal isolates were incubated at 18°C for 15 consecutive days prior to extractions.
We pooled four solid cultures of Z. tritici and extracted metabolites with 400 mL ethyl acetate (EtOAc) (Pestinorm; VWR Chemicals, Leuven, Belgium) after homogenizing by an Ultra Turrax at 19,000 rpm. Each EtOAc extract was washed twice with 200 mL of Milli-Q (Arium, Sartorius) water in a separatory funnel to remove salts. The EtOAc layer was then evaporated to dryness by a rotary evaporator (150 rpm at 40°C). Dried extracts were solubilized in methanol and filtered (0.2-μm filter) into storage vials and dried in vacuo. Each fungal strain was extracted in triplicate (biological replicates).
We pooled four solid cultures of Z. tritici and extracted metabolites with 400 mL ethyl acetate (EtOAc) (Pestinorm; VWR Chemicals, Leuven, Belgium) after homogenizing by an Ultra Turrax at 19,000 rpm. Each EtOAc extract was washed twice with 200 mL of Milli-Q (Arium, Sartorius) water in a separatory funnel to remove salts. The EtOAc layer was then evaporated to dryness by a rotary evaporator (150 rpm at 40°C). Dried extracts were solubilized in methanol and filtered (0.2-μm filter) into storage vials and dried in vacuo. Each fungal strain was extracted in triplicate (biological replicates).
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