pH Indicators-BCECF

  • #0061 BCECF (AM) 1mg...$72 Add to Cart
  • #0062 BCECF (AM) 20 x 50 ug...$105 Add to Cart
  • #0060 BCECF (Free Acid) 1mg..Discontinued

Knowledge of cytosolic pH (pHi) is essential in many cellular studies.   The most popular indicator for studying pHi is BCECF (Figure 7.5).  Its pKa of 6.98 is ideal because all cells have pHi near 7.0, with physiological changes of no more than a few tenths of a pH unit. BCECF has an excitation spectrum with a maximum at 507 nm and an isoexcitation point at 438 nm (at which the fluorescence emission is independent of pH). Intracellular pH can therefore be estimated by measuring the ratio of the fluorescence intensities at 500 and 450 nm.  We offer BCECF in the form of cell-impermeable free acid and the cell-permeable acetoxymethyl esters. (See Table 7.3)

DATA

BCECF: (2',7' -bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein)

CAS Number: 117464-70-7

Excitation: 500/450 nm (hi/low pH)

Emission: 531 nm

pKa:  7

Solubility: DMSO (AM form), H2O (salt form)

Molecular weight: 821 g/mol (AM form), 520 g/mol (salt form)

 

 

BCECF

Figure 7.5 BCECF

 

pH indicators

 

Generic loading protocol:

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.

 

 

 

REFERENCES

Kolber, M.A., et al. J. Immunol. Meth. 108, 255(1988); Carpenter, L., et al. Biochem. J. 304, 751(1994)

Weiner, I.D., et al. 1989. Am. J. Physiol. 256: F957-F964. PMID: 2719125


Miyata, H., et al. 1989. Biochem. Biophys. Res. Commun. 163: 500-505. PMID: 2775282


Hayashi, H., et al. 1992. Am. J. Physiol. 262: C628-C634. PMID: 1550207


Sasaki, S., et al. 1992. Biochim. Biophys. Acta. 1137: 45-51. PMID: 1327152


Futsaether, C.M., et al. 1993. Can. J. Microbiol. 39: 180-186. PMID: 8467419

Seller, J.R. et al. J. Immunol. Meth. 172, 255(1994)