To determine whether the cells undergo caspase dependent apoptosis upon glucose treatment, both the active form of caspase-3 and caspase-3 activity were measured in the total cell lysate using Invitrogen human active caspase-3 ELISA kit (KHO1091; Life technologies, Carlsbad, CA, USA) and (Alexis Corporation, Lausen, Switzerland), respectively, according to manufacturers’ protocol. Background fluorescence was measured in wells containing lysis buffer, assay buffer and the substrate without cell lysate and used for the normalization of the test samples. For active caspase-3 measurement readings at 450 nm were obtained using microplate spectrofluorometer (Spectra Gemini; Molecular devices, Sunnyvale, CA, USA). For caspase-3 activity, the fluorometric readings were measured at 405 nm absorption. All the measurements were carried out in triplicates and for 3 independent sets of experiments.
Spectra gemini
Manufactured by Molecular Devices
Sourced in United States
The Spectra Gemini is a microplate reader that simultaneously measures absorbance, fluorescence, and luminescence in a single microplate. It is designed to provide accurate and reliable data for a wide range of assays, including cell-based, protein, and biochemical applications.
Automatically generated - may contain errors
7 protocols using spectra gemini
1
Caspase-3 Activation in Glucose-Treated Cells
2
Intracellular ROS Quantification by DCFH-DA
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Intracellular reactive oxygen species (ROS) levels were analyzed by DCFH-DA staining [18 (link)]. Briefly, RAW 264.7 cells were seeded on a 96-well black plate at 1 × 105 cells/mL and incubated with LPS in the presence or absence of STE (10-300 μg/mL). After removing medium, 10 μM DCFH-DA in phosphate-buffered saline (PBS) was added to each well, and the plate was incubated for 30 min at 37°C. Fluorescence intensities were measured at 480 nm excitation/530 nm emission using a fluorescence microplate reader (Spectra Gemini, Molecular Devices).
3
Glucose Uptake Assay in HepG2 Cells
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Glucose uptake was evaluated using a fluorescence assay. HepG2 cells were plated at 1 × 104 cells per well in 96 well plates and incubated for 24 h. Cells were then starved for 24 h plated in serum-free DMEM containing palmitate (250 μM) and treated with various concentrations of JKW (0 - 50 μg/mL) for 24 h. Supernatants were then removed, cells were gently washed with DPBS, and media were replaced with glucose-free DMEM containing 2-NBDG (150 μg/mL). Cells were then incubated at 37°C for 20 min and rewashed with DPBS. Relative fluorescences were obtained at excitation and emission wavelengths of 485 nm and 545 nm, respectively, using a fluorescence microplate reader (Spectra Gemini, Molecular Devices). Fluorescence images were obtained using an Olympus BX50 fluorescence microscope (Olympus, Tokyo, Japan).
4
Intracellular ROS Measurement via DCFH-DA
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A fluorescent dichlorofluorescein diacetate (DCFH-DA) assay was used to access intracellular ROS concentrations. Murine macrophages were seeded on a 96-well black plate at 1 × 105 cells/mL, and incubated with LPS (1 μg/mL) in the presence or absence of SC-E3 (50, 100, 300, or 500 μg/mL). After removing medium, cells were treated with 10 μM DCFH-DA in phosphate-buffered saline (PBS) for 30 min at 37°C. Fluorescence was measured at excitation and emission wavelengths of 480 nm and 530 nm, respectively, using a fluorescence microplate reader (Spectra Gemini, Molecular Devices).
5
Cell Viability Assay with FLX
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COV434 cells were seeded in 96-well plates at 100,000 cells/well. Two days later, the medium was replaced with serum-free medium in the absence (control) or presence of FLX (0–10 µM) for 2 h or 24 h at 37°C before the addition of 20 µl of CellTiter-Blue Reagent (Promega, Madison, WI, USA) to each well. After having incubated, changes in fluorescence were recorded with a Spectra Gemini spectrofluorimeter (Molecular Devices, Sunnyvale, CA, USA) at an excitation wavelength of 560 nm and an emission wavelength of 590 nm. The fluorescent signal from the CellTiter-Blue Reagent is proportional to the number of viable cells.
6
Measuring Cytoplasmic Calcium Dynamics
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The variation in cytoplasmic Ca2+ concentration ([Ca2+]cyt) was assessed using the fluorescent Ca2+ indicator Fluo4-AM. COV434 cells were incubated with different FLX concentrations for 2 h before adding Fluo4-AM, and new incubation was carried out for 30 min at 37°C in the dark, before three washings with PBS. Changes in fluorescence were recorded with a Spectra Gemini spectrofluorimeter (Molecular Devices) at an excitation wavelength of 494 nm and an emission wavelength of 516 nm. Intracellular Ca2+ level calculations after the incubation period were based on the given dissociation constant Kd (Ca2+) = 345 nM of Fluo4-AM (Molecular Probes). The [Ca2+]cyt was calculated using the equation [Ca2+]cyt = Kd (F – Fmin) / (Fmax – F). Maximum fluorescence was determined using 50 µM calcium ionophore A23187 in the presence of 2 mM Ca2+, and minimal fluorescence was determined in the absence of Ca2+ and the presence of 2 mM EDTA.
7
Antioxidant and ROS Assays for Screening
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A DPPH assay was conducted using a slight modification of the method reported by Gyamfi et al. [29 (link)]. Reduction of the DPPH free radical was measured by reading the absorbance at 540 nm after adding each reactant. Briefly, various concentrations of samples were incubated with 50 mM Tris-HCl (pH 7.4) and 0.1 mM DPPH ethanol solution for 30 min, in a dark room.
A fluorescent 2',7'-dichlorofluorescein diacetate (DCFH-DA) assay was performed to determine the intracellular ROS concentrations. Murine macrophages were seeded on a 96-well black plate at 1 × 105 cells/ml, and incubated with LPS in the presence or absence of SC-E1. After removing the medium, 10 μM DCFH-DA in PBS was added to each well at 37 °C for 30 min. The fluorescence was measured at excitation and emission wavelength of 480 nm and 530 nm, respectively, using a fluorescence microplate reader (Spectra Gemini, Molecular Devices).
A fluorescent 2',7'-dichlorofluorescein diacetate (DCFH-DA) assay was performed to determine the intracellular ROS concentrations. Murine macrophages were seeded on a 96-well black plate at 1 × 105 cells/ml, and incubated with LPS in the presence or absence of SC-E1. After removing the medium, 10 μM DCFH-DA in PBS was added to each well at 37 °C for 30 min. The fluorescence was measured at excitation and emission wavelength of 480 nm and 530 nm, respectively, using a fluorescence microplate reader (Spectra Gemini, Molecular Devices).
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