Why is probenecid used to conduct calcium flux assays when using fluorescent calcium indicators in certain cell types?

Probenecid is an organic anion transporter inhibitor. Organic anion transporters are expressed in many cell types. They play an essential role in the excretion of numerous endogenous metabolic products and exogenous organic anions out of the cell. When intracellularly measuring calcium mobility, fluorescent calcium indicators are often used. Upon binding to intracellular calcium, the fluorescence intensity of the calcium indicator enhances significantly. This allows for the detection of calcium movement in real time.

However, after cells are loaded with calcium indicators, organic anion transporters can actively extrude the dyes back into the extracellular environment. Such transporters are particularly active in cell types such as CHO and HeLa. In these cases, probenecid is often used in tandem with calcium indicators to inhibit these transporters and improve intracellular retention of the indicators. Not only does preparing a probenecid solution add more steps to the assay procedure, it also shows apparent cell toxicity. Furthermore, probenecid changes the calcium flux kinetics of a cyclic nucleotide gated ion channel and decreases the channel’s response to agonists of GPCR. 

Because of probenecid’s negative effects, using a calcium indicator that does not require the use of probenecid is beneficial. Calbryte™ 520 AM is the first probenecid-free fluorescent calcium indicator that displays excellent intracellular retention without the use of probenecid. Calbryte™ 520 AM is cell permeable and non-fluorescent. Once inside the cell, non-specific intracellular esterases cleave off the AM esters activating Calbryte™ 520 AM. Upon binding to calcium, Calbryte™ 520 AM produces an exceptionally bright fluorescence signal that can be detected at Ex/Em = 490/525 nm.