BCECF

BCECF (CAS No.: 85138-49-4) is a widely used, cell-impermeant, pH-sensitive fluorescent probe employed for quantitative measurement of extracellular or compartmental pH in biomedical research. As a dual-excitation ratiometric indicator with a pKa of approximately 6.98, BCECF operates through protonation-dependent changes in fluorescence, enabling precise determination of pH within the physiological range of 6.0–8.0 by calculating the ratio of emission intensity at 535 nm upon excitation at 490 nm versus 440 nm. Its membrane-impermeable nature restricts localization to extracellular environments or accessible compartments unless delivered by specialized methods, distinguishing it from esterified analogs that passively diffuse into cells. BCECF is commonly applied in studies of ion transport, cellular metabolism, and microenvironmental pH regulation, particularly in models investigating acid-base homeostasis, transporter activity, and disease-associated alterations in extracellular acidity. Although it does not act on a specific molecular target in the traditional pharmacological sense, its fluorescence response is directly linked to proton concentration, making it a valuable analytical tool in signaling and metabolic pathway studies. Experimental use typically involves micromolar-range concentrations optimized according to assay conditions, instrumentation, and biological system, and it is broadly utilized in cell-based assays and physiological studies to support drug discovery efforts targeting pH-dependent processes.