Redox Cycling Compound Assay

The assay was adapted from a previously described protocol to assess redox cycling of compounds in the presence of reducing agents (51 (link)). 13 nl of compounds (or DMSO control) were pin-transferred into 2.5 μl HBSS (Thermo Fisher; containing 1.26 mm CaCl₂, 0.49 mm MgCl₂, 1 g/l d-glucose) in black 1,536 well plates. Compound fluorescence was measured immediately (READ 0) using a ViewLux uHTS microplate imager (PerkinElmer) equipped with Ex: 525/20 and Em: 598/25 filters. 2.5 μl of a 2X Amplex Red solution [100 μm Amplex Red (Cayman Chemical, Ann Arbor), 200 μm DTT (Thermo Fisher) and 2 U/ml horse radish peroxidase (Sigma-Aldrich); diluted in HBSS and protected from light] were added to each well. Fluorescence was measured after a 15 min incubation at room temperature (READ 1), using ViewLux settings identical to READ 0. Activity was calculated using corrected fluorescence values (READ 1 minus READ 0), which were compared with control samples (negative = vehicle; positive = 46 μm walrycin B).