Multimode microplate reader

The capacity of the aromatic extracts from H. coronarium to reduce ferric was evaluated through an ABTS assay modified from Saeio et al. (2011) and Chaiyana et al. (2017) [40 (link),42 (link)]. Concisely, 20 μL of the sample solutions, comprising H. coronarium aromatic extracts dissolved in DMSO at a concentration of 1 mg/mL, was mixed with 180 μL of the FRAP solution, prepared by combining 0.3 M acetate buffer pH 3.6, 10 mM TPTZ in 40 mM HCl solution, and 20 mM ferric chloride solution. The resulting mixture was incubated for 5 min at an ambient temperature. The absorbance was then measured at 595 nm utilizing a multimode microplate reader (BMG Labtech GmbH, Ortenberg, Germany). The outcomes were expressed as EC₁, calculated using the subsequent equation:
where a represented the absorbance of the combination with aromatic extracts from H. Coronarium. L-Ascorbic acid was used as the positive control. The test was performed three times.

The capacity of the aromatic extracts from H. coronarium to scavenge ABTS free radicals was evaluated through an ABTS assay modified from Tachakittirungrod et al. (2007) and Chaiyana et al. (2017) [40 (link),41 (link)]. Concisely, 20 μL of the sample solutions, comprising H. coronarium aromatic extracts dissolved in DMSO at a concentration of 1 mg/mL, was mixed with 180 μL of the ABTS solution, prepared by combining 7 mM ABTS with 2.45 mM potassium persulfate (K₂S₂O₈) and left for 16 h in the dark, followed by a dilution with ethanol to receive an absorbance of 0.7 ± 0.1 units at 750 nm. The resulting mixture was incubated for 5 min at an ambient temperature. The absorbance was then measured at 750 nm utilizing a multimode microplate reader (BMG Labtech GmbH, Ortenberg, Germany). The outcomes were expressed as the percentage of ABTS inhibition, calculated using the subsequent equation:
where a represented the absorbance of the combination without aromatic extracts from H. Coronarium and b represented the absorbance of the combination with aromatic extracts from H. Coronarium. L-Ascorbic acid was used as the positive control. The test was performed three times.

ZKM (2 × 10⁶) pre-treated with or without the indicated inhibitors were infected with A. hydrophila. The ZKM were collected at indicated time points p.i., washed, and incubated with 5 µM MitoSOX^TM Red mitochondrial superoxide indicator (Molecular Probes) for 20 min in the dark. The pellet was resuspended in 100 µL 1× PBS, and the changes in fluorescence intensity were quantified at an excitation/emission of A₅₁₀/A₅₈₀ using a multi-mode microplate reader (BMG Labtech, Ortenberg, Germany) [35 (link)].
In a parallel study, ZKM treated with MitoSOX^TM were washed, and the pellet was resuspended in 100 µL 1× PBS and incubated with DAPI (100 μg/mL, Sigma) for 15 min. The excess dye was washed and the ZKM were then mounted on a slide with fluoroshield, and three different fields were visualized using a fluorescence microscope (×40, Zeiss Imager, Z2).

Cell viability was analyzed 19–24 h (and additionally 7 days for MucilAir) post-exposure using the PrestoBlue Cell Viability Reagent (ThermoFisher Scientific, Waltham, Massachusetts). PrestoBlue assay indicates the metabolic competence of cells by measuring the reduction of resazurin to resorufin. When added to cells, the PrestoBlue reagent is modified by the reducing environment of viable cells and turns red in color and becoming highly fluorescent. This change can be detected using absorbance or fluorescence measurements. For this study, the manufacturer’s instructions were followed to assess cell viability. Briefly, after removing the basolateral medium, 1 ml of a 10% PrestoBlue solution in BEBM was added on the apical side for BEAS-2B cells or 200 µl of a 10% PrestoBlue solution in MucilAir Medium was added for MucilAir tissues. After incubation for 1 h at 37°C in the dark, fluorescence of 2 samples/well (90 µl each) from apical solution was measured in a white 96-well plate with clear bottom (Greiner, Bio-One nv) using a multimode microplate reader in fluorescence mode (excitation: 552 nm (22 nm), emission: 590 nm (20 nm), BMG Labtech, Ortenberg, Germany). Cell viability was expressed as the percentage of relative fluorescence intensity (RFI) of treated cells relative to the RFI of the N₂ control cells.

The capacity of the aromatic extracts from H. coronarium to inhibit the elastase activity was evaluated when the substrate was AAAVPN through the assay modified from Thring et al. (2009) and Laothaweerungsawat et al. (2020) [36 (link),43 (link)]. Concisely, 10 μL of the sample solutions, comprising H. coronarium aromatic extracts dissolved in DMSO at a concentration of 1 mg/mL, was mixed with 40 µL of 0.03 unit/mL elastase with the enzyme activity greater than 90%. The resulting mixture was incubated for 15 min at ambient temperature, and 100 μL of 1.6 mM AAAVPN in tris HCl buffer pH 8.0 was added to start the reaction. Immediately, the absorbance was then kinetically measured at 410 nm for 20 min utilizing a multimode microplate reader (BMG Labtech GmbH, Ortenberg, Germany). The outcomes were expressed as the percentage of elastase inhibition, calculated using the subsequent equation:
where a represented the absorbance of the combination without aromatic extracts from H. Coronarium and b represented the absorbance of the combination with aromatic extracts from H. Coronarium. Oleanolic acid was used as the positive control. The test was performed three times.