Minimax300

NF-κB activation was quantified by measuring gaussia luciferase (Gluc) reporter using the Secrete-Pair^TM Gaussia Luciferase Dual Luminescence Assay Kit (THP, Vienna, Austria; LF062) according to the manufacturer’s instructions. AP-1 activation was determined by measuring firefly luciferase (Fluc) reporter using the Luc-Pair^TM Firefly Luciferase HT Assay Kit (THP, Vienna, Austria; LF018) according to the manufacturer’s instructions. Relative luminescence units (RLU) were measured in a plate reader (SpectraMaxi3x, Molecular Devices, LLC, San Jose, CA, USA; Luminescence Glow, Lum 384 cartridge), and normalized to the cell count generated with an imaging cytometer (Mini Max 300, Molecular Devices, LLC, San Jose, CA, USA).

The relative cell proliferation rate was determined using two different methods, as follows. (1) The fluorescence intensity of turboGFP was measured over 96 h in replicates (n = 12), using the Spectramax i3x microplate reader (Molecular Devices, San Jose, CA, USA) with the Minimax 300 imaging cytometer with a wavelength 456 for excitation and 541 for emission. The relative increase in turboGFP was compared to the intensity at the first measurement (at ~15–18 h). (2) Metabolically active cells were analyzed with the CellTiter-Glo™ Luminescent Cell Viability Assay (Promega Corp., Madison, WI, USA), according to the manufacturer’s instructions. Cells were cultured in replicates (n = 6), in opaque white 96-well plates, 1 × 10⁴ and 1.5 × 10³ cells/well for the 22Rv1 and PC-3 cell lines, respectively. After 72 h, the CellTiter-Glo™ reagent (Promega Corp.) was added and luminescence was determined on the Spectramax i3x (Molecular Devices) microplate reader.