Sunrisetm microplate reader

The radical cations were prepared by mixing a 7 mM ABTS+ stock solution in water with 2.45 mM potassium persulfate and stored in the dark for 12–16 h at room temperature. The ABTS+ solution was diluted with absolute ethanol to an absorbance of 0.7 ± 0.02 at 734 nm before use. In a 96-well plate, 20 µL of each extract was mixed with an 80 µL ABTS+ solution. Measurements were taken at 734 nm using a Sunrise^TM microplate reader (Tecan Group Ltd.). To evaluate the radical scavenging activity of Compounds 1–3, each compound was dissolved in 70% ethanol (v/v) at concentrations of 0.0125–1.000 mg/mL. L-ascorbic acid in 70% ethanol (v/v) was used as a positive control at concentrations of 0.0125–0.1000 mg/mL. The ABTS+ radical scavenging activity of the DBP and DBF extracts dissolved in 70% ethanol (v/v) at concentrations of 0.125–10.000 mg/mL. The effective concentrations of the compounds or extracts required to scavenge ABTS+ radical by 50% (IC₅₀) were obtained by a linear regression analysis of the dose–response curve plotting between %inhibition and concentrations. The ABTS+ radical scavenging activity at every concentration of each compound or extract was documented in Table S2.

Serum levels of mouse chemerin were examined using enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instruction (#DY2325, Bio-Techne, USA). In brief, 100 µL diluted serum (1:500 dilution) was added as samples. Samples and standards were then incubated at room temperature for 120 min with gentle shaking. After washing three times, detection antibodies were added and incubated at room temperature for another 120 min. After washing three times, Streptavidin-HRP was added and set for 20 min at room temperature with gentle shaking. After washing five times, the substrate reagent was added in the dark with gentle shaking. After 20 min of incubation, stop solution was added, and the absorption was read at 450 nm with wavelength correction at 570 nm immediately with Sunrise^TM microplate reader (Tecan Group, Männedorf, Switzerland) and analysed by Magellan^TM software (Tecan Group, Switzerland).^{30 (link)}

The effects of miR-143 replacement on the viability of the MKN-45 cells were assessed by MTT assay. For this purpose, 8×10³ cells (vec- and vec + cells) were cultured in 96-well plates in triplicates. Then, 4 days later, the cells were treated with 50 μL of MTT solution (2 mg/mL; Lot no. DU21373R2; Bio Basic, Canada) at 37°C for 4 h. Then, 200 ml of dimethyl sulfoxide was used to dissolve the resulting formazan.^{13 (link)}
After incubation at 37°C for 30 minutes, absorbance was estimated at a test wavelength of 570 nm and a reference wavelength of 620 nm. using a Sunrise^TM microplate reader (Tecan, Switzerland).

The capacity of cellular proliferation was assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) MTT assay. Two weeks after the cell transfection, the cells (approximately 2 × 10³ cells per well) were seeded into 96-well culture plates for four days. The MTT (Sigma, Taufkirchen, Germany) at a concentration of 2 mg/ml was added to the wells and stayed for 4 h at 37 °C. Afterward, 200 µl of dimethyl sulfoxide (DMSO) was added for 30 min at 37 °C to solubilize the crystals. The absorbance was determined with a Sunrise^TM microplate reader (Tecan, Switzerland) at 570 nm (Montazami et al., 2015[27 (link)]). Each experiment was repeated three times.

Sixty percent confluent cells were released from culture flasks by trypsin, washed with PBS, and seeded into 96-well plates at 5 × 10⁴ cell/ml in culture medium. After incubation at 37 °C for various times, cell proliferation was analysed by the CellTiter 96 AQ_ueous Non-Radioactive Cell Proliferation kit according to the manufacture’s protocols. The plates were read at 492 nm with reference at 595 nm by a Sunrise^TM microplate reader (Tecan, Männedorf, Switzerland).