Luminescence Assay for Proluciferin Compounds

Proluciferin compounds (except luciferin-3FEME) and the NADPH regeneration system (20X solution A: 26 mM NADP⁺, 66 mM glucose-6-phosphate, 66 mM MgCl₂; and 100X solution B: 40 U/ml glucose-6-phosphate dehydrogenase in 5 mM sodium citrate, pH 5.5), the luciferin detection reagent (Cat# V8921), and the luciferin detection reagent with esterase (Cat# V8931) were from Promega (Madison, WI). Luciferin 6' 3-furfuryl ether methyl ester (luciferin-3FEME) was synthesized as described [11 (link)]. Triton X-100 was from Leagene (Beijing, China), and Tris-HCl was from AKZ-Biotech (Tianjin, China). Potassium chloride, ammonium bicarbonate, potassium dihydrogen phosphate, and potassium hydrogen phosphate were from Jiangtian Chemical (Tianjin, China), and 1X TBS buffer, 1X PBS (0.1 mM CaCl₂ (anhydrous), 2.7 mM KCl, 1.5 mM KH₂PO₄, 0.5 mM MgCl₂, 137 mM NaCl, 8.1 mM Na₂HPO₄, pH 7.4) buffer were from Corning (Manassas, VA). White opaque 96-well microtiter plates were from Nunc (ThermoFisher Scientific, Lagenselbold, Germany). All other chemicals and reagents used were of the highest grade available.