Name: 细胞内pH检测试剂盒 (BCECF AM)
SKU: I1508403
Availability: InStock

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文献证明

在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。

概述

细胞内pH是表征胞内氢离子浓度的核心生理指标，对细胞几乎所有生命活动均具有关键调控作用，包括细胞增殖与分化、程序性凋亡、离子跨膜转运、胞吞与胞吐作用、细胞黏附、生物酶活性、蛋白质相互作用及生物膜物质转运等生物学过程。研究表明，胞内pH的轻微异常波动即可显著改变细胞生理稳态与病理表型，其失衡与肿瘤发生发展、神经系统退行性病变等多种重大疾病的病理进程密切相关。因此，精准检测胞内pH值是细胞生物学、分子生物化学及病理生理学研究的重要技术手段。

BCECF AM是目前国际公认且应用最广泛的活细胞胞内pH比率型荧光探针。BCECF AM具有优异的细胞膜穿透能力，且自身无荧光背景，可自由穿透活细胞膜进入胞内。进入细胞后，该探针可被胞质内特异性酯酶水解裂解，脱去乙酰甲酯基团转化为带电荷的活性BCECF分子，因极性增强无法穿透细胞膜而稳定滞留于胞质内。活性BCECF分子的荧光激发光谱具有显著的pH依赖性，发射光谱相对稳定。其中，酸性微环境（低pH）下最大激发波长约为450 nm，碱性微环境（高pH）下最大激发波长约为503 nm。本产品标准检测体系推荐采用双激发单发射检测模式，优选激发波长为440 nm与505 nm，发射波长为535 nm，通过双波长荧光强度比值可有效消除细胞负载量、探针浓度、光漂白等实验误差，大幅提升检测准确性与重复性。

本产品适配荧光显微镜、激光共聚焦扫描显微镜、流式细胞仪、多功能酶标仪等主流荧光检测设备，应用场景广泛。另外，本产品配套了完整的梯度pH校准缓冲体系（pH 5.0–9.0），可精准建立pH标准曲线，从而实现胞内pH绝对值的精确定量检测。

产品组分

I1508403	组分名称	物理外观	200T	1000T	储存	单次测试使用量
I1508403A	检测缓冲液	液体	50 mL	250mL	-20℃	按需
I1508403B	BCECF AM (1000×)	液体	20 μL	100μL	-20℃，避光	按需
I1508403C	pH5.0校准缓冲液	液体	2 mL	10 mL	-20℃	100 μL
I1508403D	pH6.0校准缓冲液	液体	2 mL	10 mL	-20℃	100 μL
I1508403E	pH7.0校准缓冲液	液体	2 mL	10 mL	-20℃	100 μL
I1508403F	pH8.0校准缓冲液	液体	2 mL	10 mL	-20℃	100 μL
I1508403G	pH9.0校准缓冲液	液体	2 mL	10 mL	-20℃	100 μL
I1508403H	尼日利亚霉素 (1000×)	液体	20 μL	100μL	-20℃，避光	按需

使用方法（以96 孔板为例，酶标仪检测）

1. 细胞样品准备

将待测细胞接种于黑色96孔板内，每孔的细胞数需要控制在1000-8000个，通常宜在2000-5000个范围内，可按实验设计对细胞进行一定处理。

注：如果需要确定样品的具体pH值，就需要建立pH标准曲线，细胞接种培养时需要在实验孔的基础上多至少10个(每组2个孔)正常组的细胞（即pH标准孔）用于pH标准曲线的检测。

2. 染色工作液的配制

根据器皿类型和样品数量计算所需染色工作液的体积，使用检测缓冲液将BCECF AM (1000×)稀释为1×，此时得到的BCECF AM(1×)即染色工作液，每孔推荐使用量见下表。

器皿类型	96孔板	24孔板	12孔板	6孔板
染色工作液体积 (μL/孔)	100	200	500	1000

注1：染色工作液需现用现配，并一次性使用完毕。

注2：上述BCECF AM（1×）为染色工作液的推荐浓度，实际实验中需根据不同细胞系和实验体系进行优化，通常可在0.2×-2×摸索最佳浓度。

3. 染色

取出铺有细胞的96孔板，贴壁细胞直接吸除原有培养基，悬浮细胞需离心弃去孔内上清。每孔可加入检测缓冲液洗涤1次(选做)。每孔加入新鲜配制的染色工作液100 μL，37℃避光孵育30 min，孵育结束后可使用检测缓冲液洗涤1-3次。

注1：不同细胞染色效率存在差异，建议进行预实验确定最优孵育时间。

注2：若需要测定细胞内具体pH值，除实验孔外，pH标准孔同样需要进行本步骤染色，并在染色完成后进行步骤4的pH校准步骤；仅做组间相对pH对比可省略步骤4。

4. pH标准曲线校准（获取细胞内具体pH值必需步骤）

（1）以96孔板为例，按照每孔100μL的体积配制一系列pH5.0-9.0的pH校准工作液。均匀混合0.1μL 尼日利亚霉素 (1000×)和100μL pH校准缓冲液，即可配制成100μL pH校准工作液；

（2）ph标准孔：将完成染色的细胞孔内液体移除（悬浮细胞需离心去除），分别加入100μL不同 pH 的校准工作液，37℃避光孵育10min；

注1：pH校准工作液需现用现配，并一次性使用完毕，不可冻存。

注2：尼日利亚霉素 (1000×)可用于维持细胞内外pH稳定，pH校准工作液处理细胞10分钟后即可平衡细胞内外pH值。

5. 检测

（1）在Ex/Em=505/535nm和Ex/Em=440/535nm波长下用酶标仪进行检测(推荐顶读)，计算这两种不同激发波长下荧光发射强度的比值。

（2）以 pH 值为 X 轴、荧光强度比值为 Y 轴拟合标准曲线，将实验孔测定得到的比值代入pH标准曲线，换算得出细胞具体的pH值。

注：荧光显微镜、激光共聚焦显微镜、流式细胞仪检测可参考本试剂盒酶标仪检测步骤并结合仪器条件进行实验（对于流式细胞仪，推荐用FITC通道进行检测）

注意事项

1. 除BCECF AM和尼日利亚霉素外，其余试剂也可4℃保存。

2. 荧光染料均存在淬灭问题，请注意避光，以减缓荧光淬灭。

3. 正式检测前，建议选择 2-3 个预期差异较大的样本进行预实验。

4. 为了您的安全和健康，请穿实验服并戴一次性手套操作。

Intracellular pH is a core physiological indicator reflecting hydrogen ion concentration within cells, playing a crucial regulatory role in nearly all cellular life processes, including cell proliferation and differentiation, programmed apoptosis, ion transmembrane transport, endocytosis and exocytosis, cell adhesion, enzyme activity, protein interactions, and membrane-mediated substance transport. Studies have shown that even slight fluctuations in intracellular pH can significantly alter cellular homeostasis and pathological phenotypes, and its imbalance is closely associated with the pathogenesis of various major diseases such as cancer development and neurodegenerative disorders. Therefore, precise measurement of intracellular pH is an essential technical approach in cell biology, molecular biochemistry, and pathophysiology research.

BCECF AM is currently the internationally recognized and most widely used ratiometric fluorescent probe for measuring intracellular pH in live cells. BCECF AM exhibits excellent cell membrane permeability and has no intrinsic fluorescence background, enabling it to freely penetrate the plasma membrane and enter living cells. Once inside the cell, the probe is specifically hydrolyzed by cytoplasmic esterases, which cleave off the acetoxymethyl group to generate charged, active BCECF molecules. Due to their increased polarity, these active molecules cannot re-enter the cell membrane and remain stably retained in the cytoplasm. The excitation spectrum of active BCECF shows significant pH dependence, while its emission spectrum remains relatively stable. Specifically, the maximum excitation wavelength is approximately 450 nm under acidic conditions (low pH) and around 503 nm under alkaline conditions (high pH). This product recommends using a dual-excitation single-emission detection mode, with preferred excitation wavelengths at 440 nm and 505 nm, and an emission wavelength at 535 nm. By calculating the ratio of fluorescence intensities at two excitation wavelengths, experimental errors caused by cell loading levels, probe concentration, or photobleaching can be effectively eliminated, greatly improving detection accuracy and reproducibility.

This product is compatible with mainstream fluorescence detection instruments such as fluorescence microscopes, laser scanning confocal microscopes, flow cytometers, and multifunctional plate readers, offering broad application scenarios. Additionally, the product comes with a complete gradient pH calibration buffer system (pH 5.0–9.0), enabling accurate construction of a pH standard curve and thus achieving precise quantitative determination of intracellular pH values..

Kit Contents

I1508403	Component Name	Appearance	200T	1000T	Storage	Quantity Per Test
I1508403A	Assay buffer	Liquid	50 mL	250mL	-20℃	As needed
I1508403B	BCECF AM (1000×)	Liquid	20 μL	100μL	-20℃. Store in the dark.	As needed
I1508403C	pH5.0 Calibration Buffer	Liquid	2 mL	10 mL	-20℃	100 μL
I1508403D	pH6.0 Calibration Buffer	Liquid	2 mL	10 mL	-20℃	100 μL
I1508403E	pH7.0 Calibration Buffer	Liquid	2 mL	10 mL	-20℃	100 μL
I1508403F	pH8.0 Calibration Buffer	Liquid	2 mL	10 mL	-20℃	100 μL
I1508403G	pH9.0 Calibration Buffer	Liquid	2 mL	10 mL	-20℃	100 μL
I1508403H	Nigericin (1000×)	Liquid	20 μL	100μL	-20℃. Store in the dark.	As needed

Instructions for Use

1. Cell Sample Preparation

Seed the cells to be tested into black 96-well plates, with 1,000-8,000 cells per well typically recommended between 2,000 and 5,000 cells per well. Cells may be treated according to experimental design.

Note: To determine the exact pH value of samples, a pH standard curve must be established. During cell seeding and culture, additional wells should be included-minimum of 10 wells (2 wells per group) containing normal control cells (i.e., pH standard wells) to measure the pH standard curve.

2. Preparation of Staining Working Solution

Calculate the required volume of staining working solution based on the type of vessel and sample quantity. Dilute BCECF AM (1000×) with detection buffer to obtain a 1× solution; this 1× BCECF AM solution serves as the staining working solution. Recommended volumes per well are listed below:

Note 1: The staining working solution should be freshly prepared and used immediately.

Note 2: The above 1× BCECF AM concentration is recommended; however, optimal concentration should be optimized experimentally depending on different cell lines and experimental systems, typically ranging from 0.2× to 2×.

3. Staining

Remove the 96-well plate containing seeded cells. For adherent cells, remove the original culture medium by aspiration. For suspended cells, centrifuge and discard the supernatant. Optionally, wash each well once with detection buffer. Add 100 μL of freshly prepared staining working solution to each well, and incubate at 37 °C in the dark for 30 minutes. After incubation, wash with detection buffer 1–3 times.

Note 1: Staining efficiency may vary among different cell types; it is recommended to perform a preliminary experiment to determine the optimal incubation time.

Note 2: If measuring the intracellular pH value precisely, pH standard wells must also undergo this staining step, followed by pH calibration in Step 4 after staining. Step 4 can be omitted if only relative pH comparisons between groups are required.

4. pH Standard Curve Calibration (essential step for obtaining accurate intracellular pH values)

(1) Using a 96-well plate as an example, prepare a series of pH calibration working solutions ranging from pH 5.0 to 9.0, with 100 μL per well. Mix 0.1 μL Nigericin (1000×) uniformly with 100 μL pH calibration buffer to obtain 100 μL of pH calibration working solution.

(2) pH standard wells: Remove the liquid from the stained cell wells (for suspended cells, centrifuge and remove supernatant), then add 100 μL of pH calibration working solution with different pH values to each well, and incubate at 37 °C in the dark for 10 minutes.

Note 1: pH calibration working solutions should be prepared fresh and used immediately; do not freeze.

Note 2: Nigericin (1000×) helps maintain stable intracellular and extracellular pH; after 10 minutes of treatment with the pH calibration working solution, cellular pH reaches equilibrium.

5. Detection

(1) Measure fluorescence using a microplate reader at excitation/emission wavelengths of Ex/Em = 505/535 nm and Ex/Em = 440/535 nm (top reading is recommended). Calculate the ratio of fluorescence intensities under these two excitation wavelengths.

(2) Fit a standard curve with pH as the X-axis and fluorescence intensity ratio as the Y-axis. Substitute the measured ratio from experimental wells into the pH standard curve to calculate the corresponding intracellular pH value.

Note: For fluorescence microscopy, confocal laser scanning microscopy, or flow cytometry, refer to the microplate reader detection procedure described above and adjust according to instrument specifications (for flow cytometry, use the FITC channel for detection is recommended).

Precautions

1. All reagents except BCECF AM and Nigericin can be stored at 4°C.

2. luorescent dyes are prone to quenching; please keep them away from light to minimize fluorescence quenching.

3. Before the formal test, it is recommended to select 2-3 samples with significant expected differences for a preliminary experiment.

4. For your safety and health, please wear a lab coat and disposable gloves during the operation.

规格

规格或纯度

BioReagent, 适用于分析

英文名称

Intracellular pH Assay Kit (BCECF AM)

应用

细胞代谢

检测方法

荧光法

定量&半定量

定量

检测仪器

酶标仪

检测波长

Ex/Em=505nm/535nm，Ex/Em=440nm/535nm

敏感性

Light-sensitive

储存与运输

储存条件

-20°C储存

运输条件

超低温运输

稳定性与储存

各组分在相应的储存条件下保质期12个月。

组分与储存

I1508403	组分名称	物理外观	200T	1000T	储存	单次测试使用量
I1508403A	检测缓冲液	液体	50 mL	250mL	-20℃	按需
I1508403B	BCECF AM (1000×)	液体	20 μL	100μL	-20℃，避光	按需
I1508403C	pH5.0校准缓冲液	液体	2 mL	10 mL	-20℃	100 μL
I1508403D	pH6.0校准缓冲液	液体	2 mL	10 mL	-20℃	100 μL
I1508403E	pH7.0校准缓冲液	液体	2 mL	10 mL	-20℃	100 μL
I1508403F	pH8.0校准缓冲液	液体	2 mL	10 mL	-20℃	100 μL
I1508403G	pH9.0校准缓冲液	液体	2 mL	10 mL	-20℃	100 μL
I1508403H	尼日利亚霉素 (1000×)	液体	20 μL	100μL	-20℃，避光	按需

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技术文档和文章

i1508403_细胞内ph检测试剂盒说明书.pdf

i1508403_intracellular ph assay kit manual.pdf

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细胞内pH检测试剂盒 (BCECF AM)

储存与运输

质量文档

文献证明

概述

规格

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