Fig. 1.

An overview of the developed method, and basic spectroscopic properties of CytoRed, resorufin and bioluminescence from Emerald Luc (ELuc). (A) Schematic illustration of the CytoRed-luciferase multiplex assay. Cells expressing a luciferase reporter were plated on multi-well plates, and CytoRed viability assay and luciferase assay were sequentially performed after desired treatment of the cells. CytoRed viability assay was performed by adding CytoRed working solution that contains 1% Triton X-100 and 20 μM CytoRed after removal of the medium. Conversion of CytoRed to resorufin by cellular endogenous esterases was monitored in terms of resorufin fluorescence for 10 min, and then luciferase activity was assayed by adding D-luciferin solution. (B) Conversion of CytoRed to resorufin. (C) Absorption spectra of CytoRed and resorufin in the assay buffer. (D) Excitation and emission spectra of CytoRed and resorufin in the assay buffer. CytoRed is almost non-fluorescent, but resorufin is highly fluorescent under the assay conditions. (E) Bioluminescence spectrum of the luciferase reaction catalyzed by ELuc under the assay conditions.