Multi well plate reader

The ammonia released was estimated by the colorimetric method at 430 nm by using Nessler’s reagent as described (Gouzy et al., 2014 (link)) with modifications in a 96-well plate reader. In brief, after growth under respective stresses, samples were harvested at 3,500 rpm. The resultant supernatant was filtered by a 0.22-micron filter. To 10 μl of the supernatant, 90 μl of Nessler’s reagent was added and incubated at room temperature for 15 min. Then absorbance was measured at 430 nm in a multi-well plate reader (Biotek). For respective stress conditions, media (without inoculation) was taken as control. All the experiments were performed at least three times.

Cells were seeded in 96-well plates (1 × 10⁵ cells/well) and incubated in the presence or absence of OLA (200–1.56 μM) for 18 h. Cell viability was measured using the SRB assay. After incubation, adherent cell cultures were fixed by adding 50 μL of 50% (w/v) cold trichloroacetic acid (Sigma–Aldrich^®, St. Louis, MO, USA) and incubated for 60 min at 4 °C. The supernatant was discarded, and the plates were washed 5 times with distilled water. An amount of 50 μL of SRB (Sigma–Aldrich^®, St. Louis, MO, USA) solution (0.4% w/v) in 1% acetic acid (Panreac^®, Barcelona, Spain) was added to each well and incubated for 30 min in darkness at room temperature. Plates were washed 5 times with 1% acetic acid and air-dried. Then, 100 μL/well of 10 mmol/L Tris base, pH 10.5 (Sigma–Aldrich^®, St. Louis, MO, USA) was added and the absorbance of each well was read on a multiwell plate reader (Biotek^®, Bad Friedrichshall, Germany) at 492 nm. At last, the cell survival was measured as the percentage of absorbance compared with that obtained in non-treated cells (control cells). We also studied the toxicity of dimethyl sulfoxide (DMSO) which was used as a vehicle to dissolve OLA.

Plant extracts were evaluated for α-glucosidase inhibitory activity according to the method given by Pistia Brueggeman and Hollingsworth, 2001 [17 (link)] with slight modifications. Plant extracts (50 μL) at varying concentrations (12.5 to 400 μg/mL) was incubated with 10 μL of the α-glucosidase (maltase) ex. Yeast (Sisco Research Laboratories Pvt. Ltd.) enzyme solution (1 U/mL) for 20 min at 37 °C with an additional 125 μL of 0.1 M phosphate buffer (pH 6.8). After 20 min, the reaction was started with the addition of 20 μL of 1 M pNPG (substrate) and the mixture was incubated for 30 min. The reaction was terminated with the addition of 0.1 N of Na₂CO₃ (50 μL) and final absorbance was measured at 405 nm using Biotek multi-well plate reader. Acarbose was used as a positive control at varying concentrations (12.5 to 400 μg/mL). Enzyme activity was calculated as:

Cell viability was tested using the PrestoBlue™ assay, as described by Mossman (1983) [32 (link)]. The GBM8401 and U87MG cells were seeded into a 96-well culture plate at 3000 cells/well incubated in an atmosphere containing 5% CO₂, saturated humidity, and 37 °C for 24 h. The next day, cells were exposed to various concentrations of RTA dh404 (0, 2, 4, or 8 µM) or DMSO as a vehicle control for 24–72 h. Cells in each well were treated with the PrestoBlue reagent for at least 10 min. Finally, absorbance was measured at 560 nm (OD560) and 590 nm (OD590) using a multiwell plate reader (BioTek, Taipei, Taiwan). IC50 values were determined. All samples were assayed in triplicate, and the mean values were calculated for each experiment. The results are expressed as a percentage of the control, which was considered to be 100%. All assay results are expressed as the mean ± standard error of the mean (SEM).