BCECF is the most widely used fluorescent pH sensor, its pKa (6.98) is near physiological pH and allows the detection of cytosolic pH change with high sensitivity. The excitation spectrum of the dye undergoes a slight shift during pH change, while the wavelength of the emission maximum remains unchanged. The pH is determined ratiometrically by the relative fluorescent intensities at 535 nm when the dye is excited at 439 nm and 505 nm respectively. At low pH, BCECF is weakly fluorescent, however, fluorescence increases with increasing pH.
Generic loading protocol for BCECF:
mammalian cells can be loaded by incubation with the membrane permeant
BCECF AM. Inside the cell, nonspecific esterases hydrolyze the nonfluorescent AM ester into
the fluorescent, pH-sensitive indicator. The low
leakage rate of the negatively charged BCECF and the small intracellular
volume results in the higher intracellular concentration compared to the extral incubation concentration. BCECF is typically less susceptible to intracellular compartmentalization than calcium indicators.
1. Prepare cells in suspension
2. Dilute a1 mM aliquot of AM ester stock solution 100-500 fold into a physiological saline buffer (HBSS for example). It is recommended to use the minimum concentration of AM ester necessary to obtain an adequate signal (0.1 μM may be sufficient). The loading medium needs to be free of amino acids or buffers containing amines, to ensure there is no interference with hydrolosis of AM esters.
3. Add one-one volumes of aqueous AM ester dispersion and cell suspension. Incubate for 15–60 minutes at 4°C to 37°C.
4. Wash the cells with fresh culture medium 1-3 times.
The general protocol has also been aplied to tisue samples such as rat arteires and salivary glands, rabbit kidney and gastric glands. Typical applications involves mounting the tissue sample in a perfusion chamber and adding 1-5μM of BCECF to the perfusate for between 10-70 minutes. An wash withunmodified perfusate is then performed.
Learn more about BCECF