Chemical Structure
General Protocol (for Human Neutrophil)*Reagents:– 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)
Protocol: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
1. R. A. Steinhardt, et al., Development of K+-conductance and Membrane Potentials in Unfertilized Sea Urchin Eggs After Exposure to NH4OH. Nature. 1973;241:400-401.2. T. J. Rink, et al., Cytoplasmic pH and Free Mg2+ in Lymphocytes. J Cell Biol. 1982;95:189-196.3. A. M. Paradiso, et al., Na+ -H+ Exchange in Gastric Glands as Measured with a Cytoplasmic-trapped, Fluorescent pH Indicator. PNAS. 1984;81:7436-7440.4. S. Grinstein, et al., Phorbol Ester-induces Changes of Cytoplasmic pH in Neutrophils: Role of Exocytosis in Na+ – H+ Exchange. Am J Physiol. 1985;248:C379-C386.5. G. B. Zavoico, et al., Regulation of intracellular pH in human platelets. Effects of thrombin, A23187, and ionomycin and evidence for activation of Na+/H+ exchange and its inhibition by amiloride analogs. J Biol Chem. 1986;261:13160-13167.6. G. R. Bright, et al., Fluorescence Ratio Imaging Microscopy: Temporal and Spatial Measurements of Cytoplasmic pH. J Cell Biol. 1987;104:1019-1033.7. C. Aalkjaer, et al., Intracellular pH Regulation in Resting and Contracting Segments of Rat Mesenteric Resistance Vessels. J Physiol. 1988;402:391-410.8. K. Tsujimoto, et al., Intracellular pH of Halobacteria Can Be Determined by the Fluorescent Dye 2 E 7 Ebis(carboxyethyl)-5(6)-carboxyfluorescein. Biochem Biophys Res Commun. 1988;155:123-129.9. M. A. Kolber, et al., Measurament of Cytotoxicity by Target Cell Release and Retention of the Fluorescent Dye Bis-carboxyethylcarboxyfluorescein(BCECF). J Immunol Methods. 1988;108:255-264.10. H. Harada, et al., cAMP Activates Cl-/HCO3 – Exchange for Regulation of Intracellular pH in Renal Epithelial Cells. Biochim Biophys Acta. 1991;1092:404-407.11. C. C. Freudenrich, et al., Intracellular pH Modulates Cytosolic Free Magnesium in Cultured Chicken Heart Cells. Am J Physiol. 1992;262:C1024-C1030.12. K. Khodakhah, et al., Functional Heterogeneity of Calcium Release by Inositol Triphosphate in Single Purkinje Neurones, Cultured Cerebellar Astorocytes, and Peripheral Tissues. PNAS. 1993;90:4976-4980.13. G. Boyarsky, et al., Superiority of in vitro Over in vivo Calibrations of BCECF in Vascular Smooth Muscle Cells. FASEB J. 1996;10:1205-1212.14. S. A. Weston, et al., New Fluorescent Dyes for Lymphocyte Migration Studies Analysis by Flow Cytometry and Fluorescent Microscopy. J Immunol Methods. 1990;133:87-97.15. L. S. De Clerck, et al., Use of Fluorescent Dyes in the Determination of Adherence of Human Leucocytes to Endothelial Cells and the Effects of Fluorochromes on Cellular Function. J Immunol Methods. 1994;172:115-124.