BCECF is the most widely used intracellular pH probe. Dr. Tsien and others improved this carboxyfluorescein by introducing two extra carboxylates that allow it to be retained better by the cell. BCECF is highly water-soluble because it has 4 to 5 negative charges at neutral pH; it becomes difficult to pass through the cell membrane after loading. Its pKa value, 6.97, is higher than that of carboxyfluorescein. BCECF has an isosbestic point at 439 nm in the excitation spectra, so it can be used in ratiometry, similar to Fura 2. Wavelengths of 505 nm and 439 nm are usually used for the ratiometric assay, and 490 nm and 450 nm filters are set in front of the excitation light source. The 530 nm filter is used for its fluorescent signal. Please note that the excitation spectrum is slightly different from the absorption spectra. BCECF-AM is an acetoxymethyl ester of BCECF that enables easy loading of BCECF into cells. BCECF-AM accumulates in a cell only by incubation as do the other acetoxymethyl esters. BCECF-AM is very sensitive to moisture; it should be carefully handled. The color of the DMSO solution changes from pale yellow to dark orange with decomposition of the AM form. Therefore, hydrolysis of the AM ester can be monitored by changes in color.
Fig. 1 Cell staining mechanism
General Protocol (for Human Neutrophil)*
– 1 mM BCECF-AM/DMSO solution (1 mg BCECF in 1.45 ml DMSO)
– HEPES buffer saline (20 mM HEPES, 153 mM NaCl, 5 mM KCl, 5 mM glucose, pH 7.4)
1. Suspend cells in HEPES buffer solution to prepare 4×107 cells per ml.
2. Add 1 mM BCECF-AM/DMSO solution to the cell suspension to prepare 3 μM BCECF-AM (1/300 vol of cell suspension) as the final concentration.
3. Incubate the cell suspension at 37ºC for 30 minutes.
4. Wash the cells 3 times with HEPES buffer saline and then prepare 3×106 cells per ml of the cell suspension.
5. Determine the fluorescence intensity using a fluorescence microscope or a confocal laser microscope coupled with an image analyzer.
* Cell staining conditions depend on cell type, so it is necessary to optimize the conditions for each experiment
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Fig. 2 Cell staining with BCECF-AM
Cell type: HeLa