I’m doing calcium staining assay, but the staining efficiency is pretty low and the background is really high. I’ve seen some people add Probenecid (from MedChemExpress)—what exactly does it do? Is it necessary to add it to get better results? If anyone has experience with this, please share. Thanks a lot!
Probenecid is an inhibitor of organic anion transporters (OATs). When calcium ion fluorescent probes in the acetoxymethyl (AM) ester form (such as Fluo-4 AM or Fura-2 AM) are loaded into cells, the probes are hydrolyzed by intracellular nonspecific esterases, releasing the negatively charged fluorescent dyes (such as Fluo-4 or Fura-2), which can then bind to intracellular calcium ions.
However, certain cell lines express organic anion transporters (such as OATs or MRPs), which actively efflux the negatively charged calcium ion fluorescent probes (such as Fluo-4 or Fura-2) out of the cell, leading to a reduction in fluorescence signal. Probenecid inhibits the activity of these organic anion transporters, effectively reducing probe efflux and thereby enhancing the stability and detection sensitivity of intracellular fluorescence signals.
The answer to this question comes from MCE Technical Support.
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