384 well plate wells

The PDE inhibitory activity of PTX, R-(-)-LSF and S-(+)-LSF was evaluated using the PDE-Glo Phosphodiesterase Assay according to the manufacturer’s instruction (Promega Corporation, Madison, WI, USA). Briefly, 1,5 μl of 1× PDE-Glo reaction buffer containing 10 mU of purified hrPDE4B or hrPDE7A (SignalChem, Richmond, Canada) was pipetted into 384-well plate wells (Thermo Scientific, USA). The tested compounds were dissolved in DMSO, and a serial dilution of the inhibitors was performed using 1× PDE-Glo reaction buffer. Then, 1 μL of diluted inhibitors and 2.5 μL of cAMP solution were added to each well. After 10 min of incubation in 30 °C, 2.5 μL of PDE-Glo™ Termination Buffer and 2.5 μL of PDE-Glo™ Detection Solution were added and the plate was incubated for 20 min at room temperature. Finally, 10 μL of Kinase-Glo® Reagent was pipetted to each well and after 10 min of incubation, the luminescence was measured using a microplate luminometer (POLARstar Omega, BMG LABTECH, Ortenberg, Germany). All data points are the average of two determinations.

384-well plate wells (Thermo Scientific™, Waltham, MA, USA) were coated overnight with optimized concentration of antigens. Samples were serially diluted in ELISA diluent buffer and 15 μL of serially diluted samples were added to wells in duplicate (control vs. treated), followed by 2 h incubation at room temperature. 1.5 M NaSCN (Sigma, St. Louis, MO, USA, diluted in PBS) was added to treated wells at 20 μL/well, while ELISA diluent buffer was added to control wells. The samples were then incubated for 15 m at room temperature. Wells were washed 6x and dried. Bound Abs were detected using AP-conjugated mouse anti-human IgG MT78 (MabTech, Cincinnati, OH, USA), incubated for 2 h at room temperature. The reaction was developed using pNPP substrate (Thermo Scientific™, Waltham, MA, USA) and signal was acquired at 405 nm. Readout is expressed proportion of signal from NaSCN-treated wells over control wells of a serially diluted sample and denoted as avidity index [55 (link)].