Intracellular Cl− was measured as previously described [11 (link)]. Cells were plated in collagen-coated 96-well plates. Cells were loaded for 1 h with the Cl—sensitive fluorescent dye, N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) (Dojindo, Kumamoto, Japan) in Hank’s Balanced Salt Solution (HBBS) (110 mM NaCl, 3 mM KCl, 1.2 mM MgSO4, 1.8 mM CaCl2, 4 mM Na acetate, 1 mM Na citrate, 6 mM D-glucose, 6 mM L-alanine, 1 mM NaH2PO4, 3 mM Na2HPO4, 25 mM NaHCO3). After loading, cells were washed thrice, and fluorescence was measured on a FLUOstar OPTIMA-6 (BMG Labtech) using 350 nm excitation and 460 nm emission.
Fluostar optima 6
Manufactured by BMG Labtech
Sourced in United States
The FLUOstar OPTIMA-6 is a multi-mode microplate reader designed for fluorescence, luminescence, and absorbance measurements. It features a monochromator-based optical system and provides a flexible and configurable platform for a wide range of applications.
Automatically generated - may contain errors
4 protocols using fluostar optima 6
1
Intracellular Chloride Measurement Protocol
2
Ketamine's Impact on Mitochondrial Complexes
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The direct effect of ketamine on the electron transport chain complexes (I, II, IV and V) was measured using the MitoTox Complete OXPHOS Activity Assay Panel (Abcam, Cambridge, MA, USA) following the manufacturer’s protocol. Each of the complexes was obtained from isolated bovine heart mitochondria in their functionally active state using highly specific monoclonal antibodies attached to 96-well microplates. For each of the complexes treated with ketamine (31, 125, 500 μM), complex activity was determined by measuring the decrease in absorbance in milli-optical density per min at room temperature or 37°C, as described in the manufacturer’s protocol. Specified wavelengths (340 nm for complexes I and V, 600 nm for complex II, and 550 nm for complex IV) in kinetic mode (every min for 2 h) were used to measure the absorbance, using a FLUOstar OPTIMA-6 (BMG Labtech, Durham, NC, USA) microplate reader.
3
Assessing Endothelial Permeability with EV Treatment
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HUAEC monolayers were cultured on transwell culture insert (1 μm pore, FALCON, #353104) and treated with EVs (Control-EVs or TGF-β-EVs) for 72 h. Permeability assay was performed previously described [25 (link)–27 (link)]. Briefly, the 10 μl of 100 μg/ml FITC-dextran (70 kDa) (Merck, #46945) in the EGM-2MV medium were added into the upper well containing HUAEC monolayer followed by incubation for 30 min. To quantify the HUAEC monolayer permeability, the fluorescence intensity of FITC-dextran leaking into the lower well was measured with FLUOstar OPTIMA-6 (BMG Labtech). The obtained values were averaged and were considered as representative of leakage in HUAEC monolayers related to increased permeability to 70-kDa molecules.
4
Quantification of Hepatic Triglycerides
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Triglyceride in the liver was measured as previously described (Le Marchand et al., 1973 (link); Fujii et al., 2008 (link); Komazaki et al., 2017 (link); Sasaki et al., 2018 (link)). Briefly, the aliquots of liver lysates were added to a microcentrifuge tubes containing 37.5% KOH and heated at 70°C for 30 min. The tubes were placed in 55°C water bath overnight (n = 4). Subsequently, 50% ethanol was added, and the tubes were centrifuged. The supernatants were separated, treated with MgCl2, left on ice for 10 min, and then centrifuged again. The supernatants and a triglyceride standard (Sigma Aldrich [St. Louis, MO, USA]) were placed in a 96 well black plate with a clear flat bottom, and triglyceride levels were measured using a commercially available kit (Triglyceride quantification kit, Sigma Aldrich [St. Louis, MO, USA]). Preparation was performed by following the manufacturer’s instruction. Fluorescence intensity (λex = 540 nm/λem = 590 nm) was measured with a plate reader (BMG Labtech FLUOstar OPTIMA-6).
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