Luciferase-Specific Coelenterazine Analogues for Optical Cross Talk-Free Bioassays.

Abstract

Spectral overlaps in fluorescence (FL) and bioluminescence (BL) commonly cause optical cross talks. The present protocol introduces five different lineages of coelenterazine (CTZ) analogues, which have selectivity to a specific luciferase, and thus cross talk-free. For example, some CTZ analogues with ethynyl or styryl groups display dramatically biased BL to specific luciferases and pH by modifying the functional groups at the C-2 and C-6 positions of the imidazopyridinne backbone of CTZ. The optical cross talk-free feature is exemplified with the multiplex system, which simultaneously illuminated antiestrogenic and rapamycin activities without optical cross talks. This unique protocol contributes to specific and high-throughput BL imaging of multiple optical readouts in mammalian cells without optical contamination.