Synergy ht biotek reader

To determine oxidative enzyme activity, a 50 µL sample was mixed with 150 µL 1 mM 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) in citric phosphate buffer, pH 4, in a 96 wells plate (Greiner Bio-One, Cellstar 655180, www.gbo.com). Absorption at 420 nm was measured every min for 30 min with a spectrophotometer (Biotek Synergy HT Reader, Agilent, www.agilent.com). Activity (mM s^-1 L^-1) was calculated using the law of Lambert-Beer with an extinction coefficient of 36,000 M^-1 cm^-1.
H₂O₂ concentration was measured in 5 replicates of SMS and its tea using a hydrogen peroxide assay kit (Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit, A22188, ThermoFisher Scientific Inc, www.thermofisher.com). A spectrophotometer (Biotek Synergy HT Reader, Agilent) was used to measure the fluorescence (Em = 530/25, Ex = 590/35) and concentration was calculated using a calibration curve (0.3–5 µM) based on aqueous H₂O₂ solutions (Sigma-Aldrich). These solutions were also used to determine the stability of H₂O₂ over time.
Viability of microorganisms in SMS tea was tested by inoculating dilution series on LB medium plates. Microorganisms were grown for one week at 25 °C .

A CellTiter-Blue assay (Promega, Fitchburg, WI, USA) was used for the cell viability measurement. Briefly, hTERT RPE-1 cells were seeded in quadruplicate for the different glucose and metformin concentrations in four different 96-well microplates at a density of 5 × 10³ in 100 μL of complete DMEM F-12 medium and cultured overnight. Once the cells had adhered to the plate surface, cells were treated with varying concentrations of glucose, complete DMEM supplemented with a physiological concentration of glucose (PhG: 100 mg/dL; Gibco, New York, NY, USA, EEUU) or complete DMEM supplemented with high glucose concentrations (HG: 450 mg/dL; Gibco, New York, NY, USA, EEUU), and metformin, 1 M, 10 mM, 1 mM and 50 μM (Cayman Chemical Company, Ann Arbor, MI, USA, EEUU), and cultured for 0 h, 24 h, 48 h and 72 h. At last, 20 μL of the CellTiter-Blue reagent was added to each well at different times and incubated at 37 °C for 1 h. Then, the absorbance was measured at a wavelength of 590 nm using a Synergy|HT Biotek reader (Biotek Instruments, Winooski, VT, USA) and the All-In-One Microplate Reader Software-Gen 5 version 2.00.18 (Biotek Instruments, Winooski, VT, USA).

To evaluate the ability of lanthipeptides to induce cAMP production via GAL₁R signaling, we used the cAMP Hunter™ eXpress GAL₁R CHO-K1 GPCR Assay from Eurofins-DiscoverX. The assay was performed according to the manufacturer’s instructions. Briefly, for one assay, CHO-K1 cells expressing GAL₁R were diluted in AssayComplete™ Cell Plating 2 Reagent, and 100 µl was seeded into each well of a 96-well plate. The plate was incubated at 37 °C, 5% CO₂ in a humidified incubator. After 24 h of incubation, the media was aspirated, and 45 µl cell assay buffer/antibody mixture was added to all wells. For the inhibition of intracellular cAMP accumulation, induced by forskolin, a serial threefold dilution of the lanthipeptides (10 µM to 5 nM) in 80 µM forskolin was made. Fifteen µl of the serial dilutions was applied in duplicates to the cells. The concentration of forskolin in the assay was 20 µM. The agonists were incubated for 30 min at 37 °C. Next, 15 µL Antibody Solution and 60 μL cAMP Working Detection Solution were added, and the plate was incubated for 1 h in the dark at room temperature. Finally, 60 μL cAMP Solution A was added and, after 3 h of incubation in the dark at room temperature, the chemiluminescent signal was read with a Synergy HT Biotek reader.