Coelenterazine

Coelenterazine is a luminescent enzyme substrate for apoaequorin and Renilla luciferase. Renilla luciferase and substrate coelenterazine has been used as the bioluminescence donor in bioluminescence resonance energy transfer (BRET) to detect protein-protein interactions. Coelenterazine is a superoxide anion-sensitive chemiluminescent probe and it can also be used in chemiluminescent detection of peroxynitrite (Ex/Em = 429/466 nm)^[1][2][3]. In Vitro:Measurement of free radical production^[5].
The measurements are performed in 0.1 M potassium phosphate buffer, pH 7.0.
Coelenterazine (2.5 μM) is added to the buffer immediately prior to measurements.
Add 2 mg/mL NADH.
At room temperature using a luminometer.

HCT-8 control cells, transiently expressing Renilla luciferase (RLuc), showed low bioluminescence due to P-glycoprotein–mediated efflux transport of coelenterazine. By comparison, transiently expressing RLuc HCT-8 cells, wherein P-glycoprotein was down-regulated with shRNAi, showed high bioluminescence^[3]. In Vivo:HCC1806-RR cells modified with Rluc are xenografted into mice^[4].
Coelenterazine (2 mg/kg) is injected intravenously.
After 5 minutes, the mice are fixed and imaged for 3 to 5 minutes.
All mice demonstrated very high Rluc activity at the primary site with the majority of mice simultaneously showing metastases to inguinal ILNs^[4].