Multiskan sky microplate spectrophotometer

Beas‐2B cells were seeded at 1.77 × 10⁴ cell/well into 96‐well culture plates and incubated for 18 h. Cells were then treated with various concentrations of C6G25S (40, 20, 10, 5, and 0 μM) in triplicate for 24 h. CCK‐8 solution (10 μl) was added to each well, and cells were incubated for another 3 h. Medium only with CCK‐8 solution and medium without C6G25S served as blank and normal control, respectively. The absorbance at 450 nm was measured with a Multiskan Sky Microplate Spectrophotometer (Thermo Fisher Scientific). The relative cell viability/cytotoxicity was calculated according to the manufacturer’s instructions.

The free radical scavenging activity of HEJ30, HEJ50, HEJ70, HES30, HES50, and HES70 was determined based on their ability to react with a stable DPPH free radical following the method described by Blois [26 (link)]. The hydroethanolic extract solutions (0.49 to 500 μg/mL) were prepared and mixed with an equal volume of methanol solution of DPPH (0.03 mM). After 60 min at room temperature and protected from light, the absorbance was measured in a microplate reader (Thermo Scientific™, Multiskan™ Sky Microplate Spectrophotometer, Waltham, MA, USA) at 517 nm. The experiment was performed in triplicate. Ascorbic acid and quercetin were used as standards. The antioxidant capacity of HEJ30, HEJ50, HEJ70, HES30, HES50, and HES70 was expressed as the 50% effective concentration (EC₅₀), which was defined as the concentration (μg/mL) of extract required to reduce 50% of DPPH or to obtain 50% antioxidant effect [27 (link)].

The 17β-estradiol levels were detected by chemilluminescence. An estradiol ELISA kit (Enzo life sciences, Farmingdale, NY, USA) was used to detect the level of corresponding substances. The detailed steps were carried out according to the manufacturer’s protocol. The results were detected with Multiskan sky microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Cell viability, expressed as cell proliferation, was measured using a cell counting kit-8 (CCK-8) assay. YT cells or PBLs were added to 96-well microtiter culture plates and stimulated with different concentrations of kayadiol. At the end of each cell culture period, cells were incubated with the CCK-8 solution for an additional 2 h, and the absorbance was detected at 450 nm by a Multiskan Sky Microplate Spectrophotometer (Thermo Fisher Scientific, Eugene, OR, USA).

To check the cytotoxic potential of bacterial metabolite, commercially available TMA, on epithelial cell line HCT116 and HT29, Thiazolyl Blue Tetrazolium Bromide dye was used. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) is a colorimetric assay in which mitochondrial succinate dehydrogenase enzyme converts yellow dye into purple formazan crystals. Cell viability is directly proportional to the intensity of the purple color. Briefly, 3000/well HCT116 and HT29 cells were seeded in 96 well plate. After 24 h of incubation, cells were treated with different concentrations from 0.15 mM to 10 mM of TMA in triplicates for 24 h, 48 h, and 72 h. After incubation, 20 µl MTT dye (5mg/ml) was added to each well and the plate was incubated for 3 hours. Finally, 100 µl of dimethyl sulfoxide (DMSO) was added to dissolve formazan crystals and absorbance was recorded at 570 nm using a Multiskan sky microplate spectrophotometer (Thermo scientific) (27 (link)). Untreated cells were taken as control. To calculate % cell viability, the absorbance of untreated cells was noted as 100% viable cells.

After resazurin staining, biofilms were subsequently stained with crystal violet as previously reported [14 (link)]. To fix the biofilms, a working volume of ethanol 100% (either 40 µL or 50 µL in 384WP or 200 µL in 96WP) was added onto the biofilms and the 96WP/384WP plates were incubated at RT. After 15 min. the lids were removed, and wells were allowed to air-dry completely. The biofilms were stained with various concentrations (0.023%, 0.1%, 0.23%, 1% v/v) of crystal violet (HT90132, Sigma-Aldrich, St. Louis, MO, USA) at RT for five minutes and then washed twice with deionized H₂O and left to dry a few minutes (5 min. for 96WP, up to 10 min. for 384WP). The crystal violet bound to the biofilms was solubilized in 96% ethanol for 1 h at RT. The absorbance was measured at 595 nm with a Multiskan Sky microplate spectrophotometer (Thermo Scientific, Vantaa, Finland).