Kinase experiment:

Measurements are made at room temperature with continuous stirring of the mitochondrial suspension using spectrophotometer equipped with a magnetic stirrer with fluorescent cation R123 as probe. Excitation and emission wavelengths are 503 nm and 527 nm, respectively. The incubation medium is the respiration buffer. R123 and sodium pyruvate are added to final concentrations of 50 nM and 10 mM, respectively. Isolated mitochondria maintain a steady membrane potential (±5%) throughout the duration of the recording[1].

Rhodamine 123 is a fluorescent tracer dye used as substrate for P-glycoprotein.

Fluorescent tracer dyes represent a class of sub-cellular probes allowing the examination of cellular processes in real-time with minimal impact upon such processes. These tracer dyes are becoming increasingly used for the examination of membrane transport processes, since they are easy-to-use and cost effective probes for a number of membrane protein transporters, such as P-glycoprotein.

In vitro: Rhodamine 123, which was identified as a member of the rhodamine family of flurone dyes, was used to examine membrane transport by the ABCB1 gene product, MDR1. Previous study determined the λmax for excitation and emission for rhodamine 123 in commonly used solvents and extraction buffers, indicating that the fluorescence of rhodamine 123 was highly dependent on the chemical environment. The optimal parameters are 1% methanol in HBSS. In addition, the uptake of rhodamine 123 into cells was via both passive and active processes, and this occurred mainly by OATP1A2-mediated transport. Furthermore, this previous study quantified the intracellular sequestration and metabolism of rhodamine 123, showing that these were both cell line-dependent factors that might influence the interpretation of transport assays [1].

In vivo: So far, there is no animal in vivo data reported.

Clinical trial: Up to now, rhodamine 123 is not used clinically.

Reference:
[1] Samantha Forster et al. Characterization of Rhodamine-123 as a tracer dye for use in in vitro drug transport assays. PLoS One. 2012; 7(3): e33253.