Spectramax paradigm multi mode detection platform

A modified technique outlined by Mosmann (1983) and Vistica (1991) was used for MTT viability assay [57 (link),70 (link)]. HepG2 cells were seeded at a density of 40,000 cells/mL in a 96-well plate. The cells were left to be attached overnight, then they were exposed at different concentrations (μg/mL) of the samples. The spent medium was aspirated after 24 h of incubation at 37 °C and substituted in simple DMEM by a 0.5 mg/mL MTT. After another 3 h of incubation at 37 °C, the medium was removed and 200 μL DMSO dissolved the purple formazan crystals. A microplate reader (SpectraMax^® Paradigm^® Multi-Mode Detection Platform, Molecular Devices LLC, San Jose, CA, USA) measured the absorbance at 540 nm.

Aggregation was assessed using light scattering and ThT fluorescence. Light scattering experiments were performed on a K2 Multifrenquency Phase spectrofluorometer set at 500 nm for excitation and emission (excitation slit width 0.5 mm, emission slit width 0.5 mm). DBD samples of 2 μM in 50 mM sodium phosphate pH 7.0, 100 mM NaCl and 2 mM DTT were stirred continuously at 37 °C during the experiments. Detection of amyloid aggregates was performed by measuring ThT fluorescence at 480 nm upon excitation at 440 nm, using a SpectraMax Paradigm Multi-Mode Detection Platform (Molecular Devices) with temperature maintained at 37 °C. Time-resolved fluorescence was recorded immediately after adding 5 μM protein to pre-equilibrated buffer containing 20 μM ThT.

HEK293T (CRL-11268, ATCC) and Hela (CCL-2, ATCC) cells were cultured in high-glucose DMEM (D5796; Sigma) supplemented with 5% FBS (Gibco) and 1% penicillin–streptomycin (Fujifilm Wako Pure Chemical Corporation), at 37 °C in a humidified atmosphere containing 5% CO₂. To compare cell proliferation, WT HEK293T cells and a series of knockout cells were seeded into 96-well plates (4.0 × 10³ cells per well). Each day, a 1/10th volume of 1 mM resazurin solution in PBS was added to each well, and the cells were incubated for 3 h. The fluorescence of reduced resazurin was measured using a fluorescence microplate reader (SpectraMax Paradigm MultiMode Detection Platform; Molecular Devices) with excitation/emission at 560/590 nm.

After four weeks of adaptation, animals were randomly divided into five groups (diets) of 10 animals (five per cage), with food supplied ad libitum. Initial parameters including weight, body length and fasting glucose levels were measured. The mice were inspected for diabetes daily using test strips for rapid determination of glucose in urine (Machenery-Nagel, Duren, Germany). In addition, glucose blood testing was performed approximately every two weeks during the experiment. Mice were considered diabetic if they had a urine glucose value above 150 mg/dL and a fasting blood glucose level of over 130 mg/dL [30 (link)]. The experimental diets lasted for 72 days, after which blood insulin, glucose, and cholesterol levels were measured after overnight fasting. In addition, IFN-γ and IL-10 levels were measured [28 ] using ELISA kits (Wuhan EIAab Science, Wuhan, China) according to the manufacturer’s instructions. Standard curves were generated for each plate to determine sample concentration. Absorbance was determined using SpectraMax Paradigm multi-mode detection platform (Molecular Devices, Sunnyvale, CA, USA) and data were analyzed using GraphPad Prism software (version 6; GraphPad Software, La Jolla, CA, USA).

HuH-7 were plated at 2 x 10⁴ cells/well density on 96-well plate and incubated at 37°C and 5% CO₂ for 12 h. After that, cells were infected as described above. Cell viability was evaluated by CellTiter-Blue (Promega) according to manufacturer’s instructions. The fluorescence was measured at SpectraMax Paradigm Multi-Mode Detection Platform (Molecular Devices).