Glomax discover microplate reader

The sequence containing the wild-type (WT) binding site or the mutated (MUT) binding site was cloned into the firefly luciferase PmirGLO reporter vector (Promega Corporation). The reporter plasmid and Renilla luciferase (hRlucneo) control plasmid were co-transfected into cells in the presence of miRNA mimic or miR-NC in a 12-well plate using Lipofectamine^™ 2000 reagent. At 48 h post-transfection, the relative luciferase activities were measured using Luciferase Reporter Gene Assay System (PerkinElmer, Inc.) on GloMax^® Discover Microplate Reader (Promega Corporation).

The viability of cells was determined by an MTT assay (Sigma-Aldrich, St. Louis, MO, USA) after experiments with different concentrations of vanicoside A or vanicoside B. Dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) was used as a solvent for vanicosides. The following concentrations were used for the experiment: 2.5, 5, 10, 25, 50 and 100 µM. The MTT assay was used to estimate a mitochondrial metabolic function through the measurement of mitochondrial dehydrogenase after 24, 48 and 72 h incubation after experiments. For the experiment, the cells were seeded in 96-well microculture plates at 1 × 10⁴ cells/well. After the incubation with selected concentrations of vanicoside A or B, the experiments were conducted according to the manufacturer’s protocol. The absorbance was determined using a multi-well scanning spectrophotometer at 570 nm (GloMax^® Discover Microplate Reader, Promega, Madison, WI, USA). The mitochondrial metabolic function was expressed as a percentage of viable treated cells in relation to untreated control cells. Staurosporine was used as the cytotoxic positive control at 2 μM.

hASC-CM, derived from two different subjects, was characterized by Enzyme-Linked Immunosorbent Assay (ELISA). The assay, performed following the manufacturer’s instructions (FineTest^®, Wuhan, China), was conducted on VEGF-A (Vascular Endothelial Growth Factor A), IL-6 (Interleukin-6), HIF-1α (Hypoxia Inducible Factor-1α), and TGF-β1 (Transforming Growth Factor-β1). The protein amount was determined recording the absorbance at 450 nm using the GloMax^® Discover Microplate Reader (Promega, Milano, Italy). Each experiment was repeated 3 times and the values, expressed as ng/culture medium deriving from 1 × 10⁶ cells, were reported as mean ± S.D.
An appropriate volume of the hASC-derived protein extract, prepared as indicated in Section 2.4, was assessed by ELISA to evaluate the amount of VEGF-A, IL-6, HIF-1α, and TGF-β1. Each experiment was repeated 3 times and the values, expressed as ng/1 × 10⁶ cells, were reported as mean ± S.D.

CellTiter-Glo® Luminescent Cell Viability Assay (Promega Corporation) was used to establish cell viability which was evaluated by GloMax® Discover Microplate Reader (Promega Corporation). Cell viability was determined as the luminescence intensity relative to untreated control cells (set to 100%), and the results are presented as means ± standard errors (SEM) from three independent experiments in quadruplicates.

The enzyme solution was obtained by homogenizing normal rat feces with a microhomogenizer and centrifuging (10,000×g, 5 min, 4 °C). The enzyme solution was mixed with HST or GTE (final concentration: 0–400 μg/mL), and the β-glucuronidase substrate solution was added and incubated at 37 °C for 60 min. The fluorescence signal at the excitation wavelength of 365 nm and emission wavelength of 415–445 nm were measured by a GloMax Discover Microplate Reader (Promega, Madison, WI, USA).