Synergy mx plate reader

The activity of NcCAR variants compared to the wild-type toward three different substrates was determined monitoring NADPH depletion. The assay composition was as follows: 10 μL of NADPH (10 mM in water), 10 μL of ATP (20 mM in water), 10 μL of CAR enzyme preparation from Ni-affinity chromatography, 160 μL of MES buffer (50 mM, pH 6.0, containing 10 mM MgCl₂), and 10 μL of 100 mM substrate (cinnamic acid, piperonylic acid, or hexanoic acid) in 0.1 M KOH. Enzymes with little or no activity were used without further dilution (0.6–3.3 mg/mL). Active enzymes were used in appropriate dilutions (≥0.15 mg/mL). The depletion of NADPH was followed on a Synergy Mx Platereader (BioTek) at 340 nm and 28°C for 10 min. Blank reactions without ATP were carried out in parallel. For each protein sample, at least three separate experiments were carried out in four technical replicates, respectively.

Pre-cultures of each strain were inoculated in biological triplicates in M9 medium supplemented as described for qualitative growth screening. Following overnight growth to saturation, each culture was diluted 10-fold in deionized water to a final volume of 200 μL in a clear polystyrene 96 well plate, and the OD₆₀₀ was measured using a Synergy Mx plate reader (BioTek Instruments, Winooski, VT). Based on this path length, cells were inoculated to an initial OD₆₀₀ of 0.03 into wells of a 48-well FlowerPlate without optodes (m2p-labs GmbH, Baesweiler, Germany) containing supplemented M9 medium. The final culture volume in each well was 1.4 mL, and the final stressor concentration (when present), was the same as previously described for qualitative growth screening. Cultures were incubated in a BioLector microbioreactor system (m2p-labs GmbH) at 37 °C with 1000 rpm shaking and the light backscatter intensity was monitored.

Cells were FBS-depleted for 2 hours prior to the cAMP assays in high-glucose DMEM medium. Treatment (isoproterenol and/or compounds) was performed in high-glucose DMEM medium. Prior to assay, cells were briefly washed 1x with PBS. Subsequently, intracellular cAMP concentration was measured using a cAMP ELISA kit (ENZO, Cat.No.: ADI-901-067A), following manufacturer’s instructions. Colorimetric assay was detected by SynergyMx plate reader (BioTek).

The cytotoxic effects of the validated HTS hits and gentamicin were characterized on HEK 293T cells using the XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) cell proliferation assay (Sigma-Aldrich, St. Louis, MO). In short, HEK 293T cells maintained in Dulbecco’s minimum essential media (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (PEN-STR), and 1% GlutaMAX were seeded into a 96-well microtiter plate at 1 × 10⁴ cells/well and allowed to attach overnight for 18 h at 37°C in 5% CO₂. Cells were then treated with the HTS validated hits and gentamicin diluted in DMEM without phenol red, supplemented with 10% FBS and 1% PEN-STR, and incubated for 24 h. Cells were then rinsed with PBS, and medium was replaced with DMEM without phenol red, supplemented with 10% FBS, 1% PEN-STR, and 50 µl of XTT/PMS (1:1 ratio with cell media). Cells were incubated for an additional 3 h, and the absorbance was measured at 450 nm using a BioTek Synergy Mx plate reader. Toxicity was represented as percent cell viability relative to mock-treated growth control. Assays were performed in triplicate. Gentamicin was compared against each of the validated hits at each tested concentration in a two-way ANOVA with Sidak’s test for multiple comparisons. Differences were considered significant for P < 0.05.