BCECF, AM is a fluorescent dye that can penetrate the cell membrane to detect intracellular pH. BCECF and AM have no fluorescence. After entering the cell, they can be cleaved by the intracellular esterase to form BCECF, thereby being retained in the cell. BCECF can be excited to form green fluorescence under proper pH. The maximum excitation wavelength and emission wavelength are different due to different pH. The maximum excitation wavelength is around 503nm, and the maximum emission wavelength is around 520nm. The recommended excitation wavelength for actual detection is 488nm and emission wavelength is 535nm. BCECF and AM are not only widely used in the research of mammalian cells, but also reported to be used in the detection of intracellular pH levels in animal tissues, plant cells, bacteria and yeast. BCECF and AM are widely used in processes such as cytotoxicity, apoptosis, cell adhesion, drug resistance, and cell chemotaxis with changes in intracellular pH. When used for intracellular pH detection, the commonly used concentration of BCECF and AM is 1~10μM. BCECF-AM is a cell membrane permeable compound widely used as a fluorescent indicator for intracellular pH. Operating Instructions (for Human Neutrophil) (1) Reagent 1 mM BCECF, AM/DMSO HEPES buffer (20 mM HEPES, 153 mM NaCl, 5 mM KCl, 5 mM glucose, pH 7.4) (2) Operation ①. Prepare cell suspension with HEPES, the cell concentration is 4×107 cells/mL. ②. Add 1mM BCECF, AM/DMSO solution to the cell suspension (1/300 volume of the cell suspension), the final concentration of BCECF, AM is 3μM. ③. Incubate at 37 ºC for 30 minutes. ④. Wash the cells 3 times with HEPES buffer to make a cell suspension of 3×106 cells/mL. ⑤. Use a fluorescence microscope or a laser copolymerization microscope with an image analysis system to detect the fluorescence intensity of the cells. *The labeling conditions vary depending on the cell type. Before each experiment, please determine the best conditions. The above method is for reference only. Precautions 1) BCECF, AM may be harmful to human body, please pay attention to proper protection. 2) Fluorescent dyes have quenching problems, please try to avoid light to slow down fluorescence quenching. 3) For your safety and health, please wear lab coats and disposable gloves for operation.
