产品介绍：
BCECF, AM是一种可以穿透细胞膜检测细胞内pH的荧光染料。BCECF, AM没有荧光，进入细胞后可以被细胞内的酯酶剪切形成BCECF，从而被滞留在细胞内。BCECF在适当的pH值条件下可以被激发形成绿色荧光。最大激发波长和发射波长因pH的不同而有所不同，最大激发波长在503 nm左右，最大发射波长在520 nm左右，实际检测时推荐使用的激发波长为488 nm，发射波长为535 nm。
BCECF, AM 不仅被广泛用于哺乳动物细胞的研究，也用于动物组织、植物细胞、细菌和酵母等的体内 pH 水平检测。此外，在细胞内有 pH 变化的细胞毒性、细胞凋亡、细胞粘附、药物抵抗、细胞趋化等过程中 BCECF, AM 也被广泛应用。
BCECF在不同pH条件下的发射光谱参考下图。
实验步骤（以人中性粒细胞为例）：
1. 试剂
5 mM的BCECF, AM/DMSO
HEPES缓冲液（20 mM HEPES, 153 mM NaCl, 5 mM KCl, 5 mM glucose, pH 7.4）
2. 操作
溶液状态的BCECF, AM ester按照比例稀释成工作浓度即可，常用的工作浓度为1-10 μM。
（1）用HEPES制备细胞悬液（~10⁶ 个/mL）。
（2）将5 mM的BCECF, AM/DMSO溶液加入细胞悬液中(细胞悬液的1/1000体积)，BCECF, AM终浓度为5 μM。
（3）在37ºC培养30 min。
（4）用HEPES缓冲液清洗细胞2-3次。
（5）使用荧光显微镜或带有图像分析系统的激光共聚显微镜检测细胞的荧光强度。
备注：标记的条件因细胞种类而异，在每次实验前，请先确定最佳条件。以上方法仅供参考。
注意事项：
1. BCECF, AM可能对人体有害，请注意适当防护。
2. BCECF, AM在4℃、冰浴等较低温度情况下会凝固而粘在离心管管底、管壁或管盖内，可以20-25℃水浴温育片刻至全部融解后使用。
3. 荧光染料均存在淬灭问题，请尽量注意避光，以减缓荧光淬灭。
4. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
Product Introduction:
BCECF, AM is a fluorescent dye that can penetrate cell membranes to detect intracellular pH. BCECF, AM itself is non-fluorescent. Once inside the cell, it can be cleaved by intracellular esterases to form BCECF, which is then retained within the cell. BCECF can be excited to produce green fluorescence under appropriate pH conditions. The maximum excitation and emission wavelengths vary with pH, with the maximum excitation wavelength around 503 nm and the maximum emission wavelength around 520 nm. For practical detection, it is recommended to use an excitation wavelength of 488 nm and an emission wavelength of 535 nm.
BCECF, AM is widely used not only in the study of mammalian cells but also for detecting intracellular pH levels in animal tissues, plant cells, bacteria, and yeast. Additionally,CF BCE, AM is extensively applied in processes involving intracellular pH changes, such as cytotoxicity, apoptosis, cell adhesion, drug resistance, and chemotaxis.
Experimental Procedure (using human neutrophils as an example):
Reagents:
5 mM BCECF, AM/DMSO
HEPES buffer (20 mM HEPES, 153 mM NaCl, 5 mM KCl, 5 mM glucose, pH 7.4)
Procedure:
BCECF, AM ester in solution can be diluted to the working concentration as needed. The commonly used working concentration is 1-10 μM.
(1) Prepare a cell suspension (~10^6 cells/mL) using HEPES buffer.
(2) Add the 5 mM BCECF, AM/DMSO solution to the cell suspension (at a volume of 1/1000 of the cell suspension) to achieve a final concentration of 5 μM BCECF, AM.
(3) Incubate at 37°C for 30 minutes.
(4) Wash the cells 2-3 times with HEPES buffer.
(5) Detect the fluorescence intensity of the cells using a fluorescence microscope or a laser confocal microscope equipped with an image analysis system.
Note: The labeling conditions may vary depending on the cell type. Please determine the optimal conditions before each experiment. The above method is for reference only.
Precautions:
BCECF, AM may be harmful to humans. Please take appropriate precautions.
BCECF, AM may solidify and adhere to the bottom, walls, or cap of the centrifuge tube at low temperatures (e.g., 4°C or on ice). It can be warmed in a water bath at 20-25°C until fully dissolved before use.
Fluorescent dyes are prone to quenching. Please avoid light exposure as much as possible to slow down fluorescence quenching.
For your safety and health, please wear a lab coat and disposable gloves during the operation.
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