In vitro CV release from the ALGs was quantified by making 4 mm × 1 mm alginate + CV hydrogel disks containing 1.5 mg of CV each. Hydrogel disks were formed by injecting the partially cross-linked alginate + CV between two glass plates with a 1-mm spacer. Polymerization was completed by incubating at the fixture at RT for 30 min. Glass was removed, and then a 4-mm biopsy punch was used to create uniform disks. Disks were transferred to a 0.4-μm transwell (Millicell, #MCHT12H48) in a 12-well plate containing 3.2 ml of DMEM and incubated at 37°C with slow continuous agitation (100 rpm). Sample medium (3 ml of DMEM) was collected and fully replenished at 4 hours and 1, 2, 3, 4, 5, and 7 days. Sample medium was stored at −20°C, and CV was quantified by mass spectroscopy (Shimadzu Prominence LC-20ADXR interfaced to a 4500 QTrap mass spectrometer) using the Mass Spectroscopy Core Facility at the Zuckerberg San Francisco General Hospital under the direction of K. Lynch. T0 (initial control) gels were dissolved in DMEM to establish the baseline of CV encapsulated in hydrogel.
Prominence lc 20adxr
Manufactured by Shimadzu
The Prominence LC-20ADXR is a high-performance liquid chromatography (HPLC) system manufactured by Shimadzu. It is designed to provide reliable and accurate analysis of a wide range of samples. The system features two high-pressure pumps, a degasser, and a column oven, which work together to deliver a stable and reproducible flow of the mobile phase. The Prominence LC-20ADXR is capable of operating at pressures up to 40 MPa, making it suitable for a variety of analytical applications.
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2 protocols using prominence lc 20adxr
1
Quantifying CV Release from Alginate Hydrogels
2
Quantitative Analysis of Marine Toxins
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Perform toxin analysis according to the method described by Nishimura et al. (44 (link)). A crude toxin extract (1 mL) was taken and mixed with 60 µL of 2.5 M NaOH, followed by incubation in a water bath at 70°C for 40 min. After cooling to room temperature, 60 µL of 2.5 M HCl was added to the mixture, which was then filtered through a 0.22-µm spin filter (Pall Corporation, USA) and stored at −20°C for subsequent analysis by liquid chromatography. The reference standards for the three DSTs (OA, DTX1, and DTX2) were procured from the National Research Council of Canada’s Institute for Marine Biosciences, and absolute quantification was performed. Regression curves were constructed based on the known toxin concentrations and spectral areas, enabling the quantification of toxin levels in the samples. Analytical software was utilized to visualize the toxin spectra and facilitate quantification. The determination and quantification of LC/MS were carried out using an HPLC system (Shimadzu Prominence LC-20ADXR) coupled with a tandem mass spectrometer (4500 QTRAP LC-MS/MS system, AB Sciex Instruments, Foster City, CA). Phenomenex Kinetex XB-C18 (150 × 2.1 mm, 2.6 µm) column was utilized for toxin separation, with the mobile phase consisting of acetonitrile (solvent A) and 0.15% formic acid in water (solvent B).
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