Multiskan go reader

Blood samples were obtained from the abdominal aorta of each mouse; the sera were separated using a BD Microtainer Blood Collection Tube (BD Life Sciences, Franklin Lakes, NJ, USA) and used to measure activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH). ALT and AST were measured using the protocols of Reitman and Frankel [17 (link)], and LDH was measured using a commercial kit purchased from Dogen (Seoul, Korea). The results were quantified with a spectrophotometer using a Multiskan GO reader (Thermo Scientific, Waltham, MA, USA).

The cytotoxicity of hit compounds was assessed by a CCK-8 assay in Caco-2 cells. At 37°C, Caco-2 cells (1 × 10⁵ cells/mL) were seeded in a 96-well plate for 24 h. Further, different concentrations of hit compounds were added to 96-well plates at final concentrations of 6.25, 12.5, 25, 50, and 100 μM. After incubation for 24 h, the plate was incubated at 37°C for 1 h with 10 μL of CCK-8 (MCE, China). A Multiskan Go Reader (Thermo Fisher Scientific) was used to measure the absorbance at 450 nm.

Hepatic lipid peroxidation was measured using the thiobarbituric acid reactive substrate (TBARS) assay. The following experiment was performed in accordance with the Volpi and Tarugi procedures [26 (link)]. The liver was homogenized in a triple volume of 1.15% KCl. The liver lysate was mixed with 6.7% trichloroacetic acid (TCA) for 15 min on ice and centrifuged at 10,000× g for 15 min at 4 °C. The supernatant was mixed with an equal volume of 0.67% thiobarbituric acid (TBA). Then, the mixture was incubated at 100 °C for 10 min. The complex levels of lipid peroxidation and TBA were measured colorimetrically at 532 nm using a MULTISKAN GO reader (Thermo Scientific).

Cytotoxicity of GSH was assessed using an MTT assay and annexin V-FITC staining. In brief, HaCaT cells (cell number: 1 × 10⁴) were treated with 0.1, 0.25, 0.5, and 1.0 mg/mL of GSH in DMEM media. After 24, 48, and 72 h of treatment, 50 µL of MTT solution was added to the cell and incubated for 1 h before aspirating the medium. After that, 100 µL of dimethyl sulfoxide (DMSO) was added and the percentages of viable cells were calculated by measuring the absorbance of 540 nm using a MULTISKAN GO reader (Thermo Scientific, Waltham, MA, USA). Apoptosis was measured by simultaneous staining with annexin V-FITC and propidium iodide (PI). HaCaT cells (1 × 10⁶) were treated with 0.1, 0.25, 0.5, and 1.0 mg/mL of GSH. After 72 h treatment, cells were harvested, trypsinized, washed with cold PBS, and stained with 1X binding buffer containing annexin V-FITC solution and PI. Subsequently, cells were incubated at room temperature for 15 min in the dark. The stained cells were analyzed by flow cytometry within 1 h. Both apoptotic and live cells were analyzed using a Becton Dickinson FACSscan flow cytometer and BD FACSDiva software (BD Biosciences, San Jose, CA, USA).

Cell cytotoxicity was measured using the DG-LDH500 kit (DoGenBio, Seoul, Korea). The cells were grown to 90% confluency, treated with different concentrations of BAC for 30 min. The 10 μL supernatant was collected and mixed with LDH Reaction Mixture (100 μL). After incubation for 30 min at room temperature (RT), the absorbance was measured at 450 nm using MULTISKAN GO reader (Thermo Scientific).

The crystal violet assay used in this study was carried out in accordance with a study published previously^{39 (link)}. Briefly, disks with multispecies biofilms after 48 h were washed twice with PBS and fixed for 15 minutes using 99% methanol. Then, the well contents were aspirated and the plates were allowed to dry. The biofilms were stained with 1 mL 0.1% crystal violet solution for 5 min. Excess stain was gently rinsed off with tap water and the plates were dried thereafter. The obtained stain was resolubilized in 1 mL of 95% ethanol with shaking in an orbital shaker for 30 min, and the ethanol was transferred to a new 96-well-plate. The solution absorbance was then measured using a Thermo Scientific Multiskan GO reader (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 595 nm.