Melanocytes were harvested and rinsed with PBS. For the assay of total alkali melanin, 1 ml of 0.2 M NaOH was added, and the absorbance of the solution was measured spectrophotometrically at 475 nm. For the assay of eumelanin, the cells were hydrolyzed in hot 30% hypophosphoric acid and hydriodic acid. After cooling, 50% ethanol was added, and the cell samples were centrifuged at 2234 g in a Model 4225 centrifuge (Associated Clinical Laboratories, Chicago, IL, USA; no longer manufactured) for 10 min. Insoluble eumelanic pigments were selectively solubilized in hot sodium hydroxide and hydrogen peroxide and the solution was cleared by centrifugation at 10,700 g for 1 min in a Sorvall Ultracentrifuge (Thermo Fisher Scientific). The absorbance of the supernatant was measured at 350 nm. For the assay of pheomelanin, the cells were solubilized in phosphate buffer (pH 10.5) and cleared by centrifugation at 10,700 g for 10 min before determination of the absorbance at 400 nm. The melanin levels were normalized to the total number of cells. All experiments were performed in triplicate.
Sorvall ultracentrifuge
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Sorvall ultracentrifuge is a high-speed centrifugation instrument designed for the separation and purification of molecular and cellular components. It utilizes powerful centrifugal forces to efficiently fractionate complex biological samples, enabling researchers to isolate and study specific biomolecules, organelles, or cells.
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Lab products found in correlation
4 protocols using sorvall ultracentrifuge
1
Melanin Quantification in Cells
2
Mineral Specimen Purification and Fractionation
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The four mineral types chosen for this study were goethite, kaolinite (City College of New York Rock and Mineral Collection); illite, and montmorillonite (Wards Science, Rochester, NY). Composition and purity of mineral specimens was confirmed by X-ray powder diffraction (City College of New York). All sediments were first crushed into powders and each washed with a 5% sodium hypochlorite (bleach) solution overnight to remove any possible organic matter or microbial contaminants. Sediments were then washed multiple times in distilled water to remove the sodium hypochlorite residue. The small clay fraction (<0.2 μm) was separated by centrifugation in a Sorvall ultracentrifuge (ThermoFisher Scientific, Waltham, MA) with an SS34 rotor at 5000 rpm (3000 g) for 1440 s (montmorillonite), 1140 s (illite), 1200 s (kaolinite) or 720 s (goethite). The supernatants containing the small fraction were collected and sterilized by autoclaving at 121 °C and 100 kPa above atmospheric pressure for 1800 s to eliminate possible bacterial contamination.
3
Isolation of Exosomes from HCT116 Cell Culture
4
Exosome Isolation via Ultracentrifugation
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Ultracentrifugation was used for two reasons: 1) characterization of exosome secretion from cells in each well plate and 2) ExoBead device characterization. In both cases, a Sorvall ultracentrifuge (ThermoFisher, USA) was used. For comparison study, the same volume of initial plasma sample was used but diluted into 1 mL of PBS. Blood samples were first centrifuged at 2000 g for 15 min, and then 12 000 g for 20 min. After initial ultracentrifugation at 100 000 g for 90 min, the supernatant was aspirated and another 38 mL of PBS was injected for a second round of ultracentrifugation at the same conditions. The pellet after the second UC was gently spiked into 500 or 200 µL, for well‐plate samples and ExoBead characterization samples, respectively.
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