Fluoforte ca2 assay kit

A FluoForte Ca²⁺ assay kit (Enz 51,017, Enzo Life Sciences, Farmingdale, NY, USA) was used for monitoring intracellular cytosolic free Ca²⁺ over time after inhibition of CK2 activity. PC3-LN4 (10 × 10³), C4–2B and 22Rv1 (15 × 10³, each) cells were plated in 100 μL total volume per well onto black-walled clear bottom 96-well tissue culture plates and grown overnight. When cells reached 70–80% confluency, they were loaded with Fluoforte dye which had been prepared in kit assay buffer [Hanks’ buffer with 20 mM HEPES (HHBS)] containing dye efflux inhibitor, and incubated for 45 min (min) at 37 °C. The dye charged cells were treated with CK2 inhibitor drugs which had been prepared in kit assay buffer; (TBB from 8 to 80 μM, and CX-4945 at 10 μM); DMSO was added for baseline negative control and ATP (150 μM) for positive control. Ca⁺² changes were detected by recording fluorescent signals at excitation 490 nm/emission 525 nm with fluorometric plate reader (SpectraMax, Molecular Devices, San Jose, Ca, USA) in kinetic mode for 15 min.