产品说明
DescriptionApplicationReferencesDataS.D.SProduct DescriptionBCECF 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 mechanismGeneral 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 experiment1. 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.Fig. 2 Cell staining with BCECF-AMCell type: HeLaRelated Categories Intracellular Fluorescent Probes
Dojindo细胞分析
细胞活力和细胞毒性测定用于药物筛选和化学物质的细胞毒性测试。Dojindo开发了高度水溶性的四唑盐，称为WST。WST-8是高度稳定的WST，用于Cell Counting Kit-8（CCK-8）。由于WST-8甲maz是水溶性的，因此不会形成晶体。因此，不需要诸如MTT测定的增溶过程。此外，CCK-8的检测灵敏度高于其他四唑盐，例如MTT，XTT，MTS或WST-1。
细胞增殖/细胞毒性转染细胞染色细胞内荧光探针细菌染色微生物活力测定干细胞分化SPiDER-ßGal线粒体检测细胞代谢
| 应用 | 产品展示 |
| 细胞生长检测，药物筛选，比色/荧光检测 | 细胞计数试剂盒-8 细胞计数试剂盒8 + 96孔有机硅定向剂 细胞计数试剂盒-F 细胞毒性LDH检测试剂盒 -WST 96孔有机硅定向剂 MTT |
| 了解检测机制的差异： | 点击这里 |
| 细胞周期分析 | 细胞周期测定溶液深红色 细胞周期测定溶液蓝色 |
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