Sense microplate reader

This assay was conducted following the manufacturer’s instructions (Promega, Fitchburg, WI, USA, cat. no. G6320). In short, 10,000 cells/well of WM9, WM164, and MUG-Mel2 cells were seeded in 96-well plates (100 µL, white, flat bottom) and treated with various concentrations of 6 for 6 h, 24 h, and 48 h. Staurosporine (10.0 µM) served as positive control for apoptosis induction. Afterwards, 20 µL of a freshly prepared viability/cytotoxicity reagent was added to each well, briefly mixed for 30 sec by orbital shaking (300–500 rpm), and incubated for 30 min at 37 °C. Fluorescence was measured at 400_EX/505_Em (viability) and 485_EX/520_Em (cytotoxicity) by using a Hidex Sense Microplate Reader. Subsequently, 100 µL of a freshly prepared Caspase-Glo^® 3/7 reagent was added to each well, briefly mixed by orbital shaking (300–500 rpm, 30 s), and incubated at room temperature for another 30 min. Luminescence was measured using a Hidex Sense Microplate Reader. Each assay was performed at least two times, with three replicates each.

The ApoToxGlo^TM Triplex Assay was purchased from Promega (Fitchburg, WI, USA, cat. no. G6320) and performed according to the manufacturer’s instructions. In brief, 10,000 cells/well (WM9 or MUG-Mel2 cells) in 100 µL of medium were pipetted in 96-well plates (white plates, flat bottom), incubated for 24 h and then treated with different concentrations of 5 for 4 h, 24 h and 48 h. Staurosporine (Abcam, Cambridge, UK) at a concentration of 25.0 µM is known to induce apoptosis and served as positive control. Subsequently, the viability/cytotoxicity reagent was prepared and 20 µL added to each well. After mixing for 30 s (orbital shaking, 300–500 rpm), the plates were incubated for another 30 min at 37 °C. Fluorescence was then measured at 400_Ex/505_Em (viability) and 485_Ex/520_Em (cytotoxicity) using a Hidex Sense Microplate Reader. Afterwards, the Caspase-Glo^® 3/7 reagent was prepared and 100 µ were added to each well, followed by mixing (orbital shaking, 300–500 rpm, 30 s), and incubation at room temperature for another 30 min. Finally, luminescence was measured using a Hidex Sense Microplate Reader. The assay was performed at least two times, with three replicates each.

The activity of β-glucuronidase enzyme and the amount of ammoniacal nitrogen were measured from fecal samples to evaluate differences in these potentially harmful microbial metabolic activities. Ammoniacal nitrogen assay was carried out by an indophenol blue method reported in detail elsewhere [21 (link)]. Briefly, 0.1 g of wet fecal sample was diluted with 5 mL of deionized water, shaken for 60 min, and centrifuged at 3000× g for 3 min. Ammoniacal nitrogen concentration was measured from supernatant based on absorbance measured at 630 nm (Hidex Sense microplate reader, Hidex Oy, Turku, Finland). β-glucuronidase assay was carried out by the protocol of Shen [22 (link)]. Briefly, 0.1 g of wet fecal sample was diluted with 5 mL of deionized water and shaken for 60 min. 0.1 mL of diluted sample was added into Eppendorf tube^® with 0.4 mL of 2 mM p-nitrophenyl-β-d-glucuronide solution (Sigma Aldrich, WGK Germany). Suspensions were incubated in anaerobic conditions at 37 °C for 60 min, followed by addition of 0.5 mL of 0.5 M NaOH. This suspension was centrifuged at 3200× g for 10 min and absorbance was measured on 405 nm (Hidex Sense microplate reader, Hidex Oy, Turku, Finland).