White 96 well plate

Recombinant SseK1, SseK2 and point mutant proteins were prepared as described and GT kinetics were measured using UDP-Glo^TM Glycosyltransferase Assay kit (Promega, #V6961) by manufacturer’s instruction. Enzyme reaction buffer (ERB) was prepared as 25 mM Tris-HCl [pH 7.5], 50 mM NaCl, 4 mM MnCl₂, 1 mM DTT and reaction was eliminated by using the nucleotide detection buffer. For preparing the acceptor-substrate, L-arginine (Duchefa BIOCHEMIE, > 98.5% purity) was dissolved in ERB. Synthetic peptides of GAPDH (Genscript, > 90% purify) was purchased and dissolved in ERB. White 96-well plates (ThermoScientific) were used for luminescence assay and the plate was read by using luminometer (VictorX5, PerkinElmer). Kinetics parameters were calculated using GraphPad Prism5 ver.5.03 software. The points represent an average of two samples and error bars represent mean ± S.D. The final specific activity of the transfer reaction was corrected considering the hydrolysis reaction, which was performed using SseK1/SseK2, UDP-GlcNAc, and MnCl₂.

Aliquots of 100 μL from the aggregation assays were collected at the indicated time points and added to white 96-well plates (Thermo Scientific, Waltham, MA USA) with 74 μM thioflavin-T solution, allowing incubation for 5 min in the dark. Fluorescence was measured using a SpectraMax i3X (Molecular Devices, San Jose, CA, USA) at 445 nm excitation and 485 nm emission.

Activation of various GIPR constructs was assessed by a luciferase reporter gene assay using a previously described protocol (Al-Sabah et al., 2014 (link)). Briefly, Flp-In HEK-293 cells were transiently transfected with cDNA encoding either GLP-1 or GIP receptor constructs and a reporter gene construct consisting of a cAMP-response element fused to a reporter gene encoding firefly luciferase (Cre-luc) using Effectene (Qiagen, Hilden, Germany), following the manufacturer’s protocol. Twenty-four hours after transfection, the cells were seeded into white 96-well plates (Thermo Scientific, Roskilde, Denmark) at a density of 10,000 cells/well. Twenty-four hours later, the cells were incubated for 3 h in media containing peptide ligand and then lysed. Luciferase activity was quantified using LucLite reagent (PerkinElmer Life and Analytic Sciences, Wellesley, MA, USA).

The intracellular levels of reactive oxygen species (ROS) were detected by DCFH-DA (2ʹ,7ʹ-Dichlorofluorescin Diacetate; Sigma-Aldrich, St. Louis, MO, USA). The cells were seeded in white 96-well plates (Thermo Fisher Scientific, Nunc A/S, Roskilde, Denmark) at a density of 1×10⁴ cells per well and maintained in 200 μL of the previously described media formulations (NC1-NC4), supplemented by 10% FBS. After growing for 48 hours, the cells were incubated with 20 μL of 100 μM DCFH-DA, which was added to the culture media. After 45 min of incubation, the medium containing DCFH-DA was replaced with 200 μL of fresh medium. The fluorescence intensity was measured immediately (zero point) and after one hour, on a plate reader Infinite 200 PRO (Tecan Group Ltd., Männedorf, Switzerland). The excitation/emission wavelengths for DCFH-DA were set at 500/529 nm. The values of the emitted fluorescence were expressed as arbitrary units, which represent the difference between the two points of measurement (one hour and zero point). Additionally, the values were corrected with respect to the cell numbers, which varied in relation to the treatment applied.

WT and myc-tagged GLP-1R activation was assessed using a luciferase reporter gene assay, as described previously [35 (link)]. Briefly, HEK-293 cells were transiently transfected with cDNA encoding GLP-1R and a reporter gene construct consisting of a cAMP-response element fused to a reporter gene encoding firefly luciferase (Cre-luc) at a ratio of 1:2. Twenty-four hours after transfection, the cells were seeded into white 96-well plates (Thermo Scientific, Roskilde, Denmark) at a density of 10,000 cells/well. Twenty-four hours later, the cells were incubated for 3 h in medium containing GLP-1 and then lysed. Luciferase activity was quantified using LucLite reagent (PerkinElmer Life and Analytic Sciences, Wellesley, MA, USA).

To determine AI-2's role in inducing effect, AI-2 inhibitor D-ribose (0, 200, 300, and 400 mM) was added to the co-culture system; then, the plantaricin production of these samples at 24 h was detected according to Section 2.2, as described earlier. The plantaricin production in co-culture was used as the positive control, and that in mono-culture was used as the negative control.
The AI-2 activity was detected by the bioluminescence of Vibrio harveyi BB170. After overnight culture at 30°C, V. harveyi BB170 was diluted in a ratio of 1:5,000 with fresh AB medium. The CFSs of the above samples were adjusted to pH 7.0, then filtrated with a 0.22-μm sterile filter, and added to the diluted BB170 culture at the percentage of 10%. The mixture was incubated at 30°C for 4 h under aerobic conditions (180 rpm), and 200 μl of aliquots were added to white 96-well plates (Thermo, USA) to measure relative luminescence units (RLUs) using the Multi-Detection Plate Reader (SpectraMax i3, Molecular Devices, USA). The suspension of strain BB170 in AB medium (1:5,000) was used as a blank control.