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A Protocol for Measurement of Intracellular pH

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Molecular Cell
Jan 2013



Intracellular pH (pHi) is an important physiological determinant of enzyme activity and cellular function (Kurkdjian and Guern, 1989). All proteins depend on a tightly regulated pH to maintain their structure and function. Protonation–deprotonation events can dictate the charge of biological surfaces and are integral steps in many metabolic reactions (Casey et al., 2010). Moreover, the proton gradient across the mitochondrial membrane is used to generate cellular energy and support other mitochondrial processes. As a result, cells have developed multiple mechanisms to maintain a narrow range of pHi in response to extra- and intracellular fluctuations in pH (Orij et al., 2012). Here, we describe a protocol for pHi measurement in live cells that uses fluorescent microscopy and the pH sensitive dye 2’,7’-Bis-(2-Carboxyethyl)-5-(and-6-)-Carboxyfluorescein Acetoxymethyl Ester (BCECF-AM). This method was recently used to determine the effects of intracellular pH changes on global histone acetylation levels (McBrian et al., 2013).

Materials and Reagents

  1. Hela cells
  2. Nigericin (Sigma-Aldrich, catalog number: N7143 )
    Prepare 10 mM nigericin (1:10,000 ) stock in ethanol (store aliquots at -20 °C and keep on ice during experiment)
  3. DMSO (Sigma-Aldrich, catalog number: D2650 )
  4. BCECF-AM (Life Technologies, catalog number: B1170 )
  5. Glucose
  6. 1% Antibiotic-Antimycotic (Gibco®, catalog number: 15240 )
  7. DMEM (Mediatech, Cellgro®, catalog number: 10-013-CV )
  8. 10% FBS
  9. Paraffin wax
  10. Calibration solution (see Recipes)
  11. 10x Earle’s balanced salt solution (EBSS) stock without glucose and without sodium bicarbonate (see Recipes)
  12. Loading solution (see Recipes)
  13. Cell culture medium (see Recipes)


  1. Perfusion inserts for 35 mm Dishes (Warner Instruments, catalog number: RC-33DM )
  2. 35 mm poly-lysine coated glass bottom culture dishes (MatTek, catalog number: P35GC-1.0-14-C )
  3. 50 ml conical tube
  4. Needles
  5. Aluminum foil
  6. Axiovert 200 M Zeiss florescent microscope equipped with a high-resolution video camera (ZEISS, Axio CAM MRm)
  7. BCECF filter set (Chroma Technology Corporation, catalog number: 71001a )
  8. Neutralize density filter (ND 1.0 A - 10.0% transmission-25mm; this is custom-designed for a given microscope) (Chroma Technology Corporation)
  9. Tubing and connections for perfusion
  10. Flow pump (Rainin Peristaltic pump Dynamax RP-1. 4-channel; catalog number is different based on supplier)
  11. 5% CO2 tank
  12. 37 °C Water bath
  13. 5% CO2 incubator (make sure it is calibrated regularly; variations in CO2 concentration can have dramatic effects on pH, generating noise in the experiment)


  1. AxioVision 4.8
  2. Slidebook 4.2
  3. SigmaPlot
  4. Excel or any spreadsheet analysis software


  1. Grow cells (~105) in 35 mm glass bottom dishes to 40% confluency.
    Note that in comparison to plastic dishes it takes longer for cells to attach to glass bottom dishes.
  2. Treat cells with drug of interest or siRNA for the desired length of time up to when cells reach ~40% confluency.
  3. A perfusion system should be installed to reduce background signal due to BCECF leaking out of the cell and to keep media temperature and pH constant throughout pHi measurement.
    1. Outflow and inflow tubes should have the same length to prevent overflow or drying of the plate.
    2. 2 ml/min is the recommended rate of perfusion; faster rates may have a shearing effect on the cells which affect the pHi.
    3. To connect the outflow and inflow tubes to the dish, we used needles that we bent into the shape of hooks and fixed them to the dish with paraffin wax. Premade sets are available commercially: https://www.warneronline.com/product_info.cfm?id=958.
    4. For efficient flow and to prevent turbulence, we used perfusion inserts for 35 mm dishes and fixed them in the dish with paraffin wax. We recommend testing the smoothness of the flow by a visible dye such as trypan blue to confirm efficient flow before the start of experiment.
    5. Wash perfusion system with distilled water for 4 h prior to the experiment to remove any residue - especially nigericin from a previous experiment – inside tubes.
  4. Set up a rack inside a water bath at 37 °C to hold a 50 ml conical tube containing the perfusion solution which is the same as the loading solution. Bubble 5% CO2 at slow to moderate rates (~1 bubble/sec) for at least 20 min into the loading solution prior the experiment to equilibrate pH, similar to the conditions inside an incubator. Continue bubbling 5% CO2 into the loading solution during the experiment to maintain pH.
  5. Have a calibrated pH meter handy. The pH of the loading solution can be continuously monitored to ensure consistency.
  6. Set up the perfusion system and the settings on the microscope. Install the Chroma filter-set for BCECF and define the channels for the software. Check the filter wheel and make sure the excitation and emission channels are defined correctly in the Axiovision software.
  7. Make sure bulb intensity is at 50% and filter density at 10% to prevent phototoxicity to the cells.
  8. Open Axiovision and set exposure times to 100 msec for 440 nm and 200 msec for 495 nm. Emission is set at 535 nm. Also specify the program to take 6 pictures at 30 sec interval.
  9. Dilute a vial of BCECF in 80 µl DMSO. Use pre-aliquotted BCECF powder to prevent repeated freezing and thawing cycles (the BCECF dye does not work well after 2 freeze-thaw cycles).
  10. Remove media from cells and wash cells with 3 ml of loading solution to remove residual media (certain amino acids interfere with dye loading).
  11. Pipet 2 ml 1x EBSS onto the plate of cells followed by addition of 10 µl of BCECF directly onto the plate. Mix gently by pipetting the EBSS-BCECF solution up and down.  
  12. Place cells in the incubator for 25 min. Start flowing loading solution through the perfusion system 5 min before the incubation is done.
  13. To prevent photo-bleaching, wrap the cell plate with a piece of aluminum foil as soon as it is removed from the incubator and is being transferred to the microscope room. Turn off the lights in the room (the light from the computer screen should provide ample light).
  14. (Work fast in this step) Immediately mount the loaded dish on the microscope stage and fix it in place with paraffin wax to prevent any movement during image acquisition. Install perfusion insert and inflow and outflow tubes. Run the perfusion system and meanwhile focus the microscope to find a suitable area – about 40% confluent – for taking pictures. We recommend using the halogen light at the weakest possible intensity for focusing and then using fluorescent light for fine focusing to reduce the risk of cell toxicity. 10x–40x magnification could be used.  With 10x magnification larger number of cells can be evaluated.
  15. Let loading solution run through the system for 2-3 min and then take 6 pictures at 30 sec intervals (automated at step 6) (Figure 1).

    Figure 1.  The final set up for intracellular pH measurement is shown. The components of the system are labeled.

  16. Import the pictures to Slidebook (make sure you define the channels correctly in Slidebook).
  17. After importing the pictures, define the background by selecting an area close to the middle of the picture with some cells nearby. Then select the cells using the Mask tool. In the Ratio tool, define 495 as the numerator and 450 as the denominator. Extract the ratio data of 495/450 channels and import the data to Excel. Remove outliers. Convert the ratio data to pH values using the calibration equation (see below).

In situ calibration

  1. Intracellular pH measurements with BCECF are made by determining the ratio of emission intensity at 535 nm when the dye is excited at ~490 nm (pH-dependent) versus the emission intensity when the dye is excited at its isosbestic point of ~440 nm (non pH-dependent). In situ calibration of BCECF's fluorescence is done the same way but in presence of 10–50 µM nigericin and 100–150 mM K+ to equilibrate internal and external pH. In situ calibration should be done for every experiment.
  2. After pictures are taken, aspirate the loading solution carefully from the loaded and fixed plate on the microscope and add 2 ml of calibration solution slowly using the side of the plate. Aspirate the calibration solution and then slowly add 2 ml of calibration solution with nigericin using a pipette. Wait 5 min before taking 2 pictures. Repeat this process at pH values 5.5, 6, 6.5, 7, 7.5, 8 and 8.5. No flow is needed at this point.
  3. Import pictures to Slidebook and analyze as described above to obtain the 495/450 ratio for each calibration pH value.
  4. To convert the ratios to pH, use SigmaPlot to obtain an equation that best describes the data. The calibration data should fit a sigmoidal plot. Use the equation that describes the sigmoidal plot to convert the experimental ratios obtained above to pH values (Figure 2).

    Figure 2.  A typical sigmoidal calibration curve and the formula describing it are shown 


  1. Calibration solution
    135 mM KCl
    2 mM K2HPO2
    20 mM HEPES
    1.2 mM CaCl2
    0.8 mM MgSO4
    adjust pH to 5.5, 6, 6.5, 7, 7.5, 8, 8.5 by adding HCl or KOH
  2. 10x EBSS stock (without glucose and without sodium bicarbonate)
    2.65 g/L
    MgSO4 (anhydrous)
    0.9767 g/L
    4 g/L
    68 g/L
    0.122 g/L
  3. Loading solution
    1x (from 10x stock)
    1 g/L (from 100 g/L stock)
    24 mM (for pH 7.4)
    1. The amount of NaHCO3 needed is determined by the desired pH of the loading solution.
    2. Adjust the osmolarity of the solution to 290-310 mOsm by adding gluconate or sorbitol.
    3. Prepare enough solution to have about 25 ml per plate.  Filter the solution and aliquot into 50 ml conical tubes. Place the tubes with their caps slightly open in a 5% CO2 incubator overnight for pH equilibration.
  4. Cell culture medium
    Note: Hela cells were cultured in 10 cm dishes using the following cell culture medium.
    Cellgro DMEM
    10% FBS
    1% Antibiotic-Antimycotic
    5% CO2, 37 °C incubator


This protocol has been modified from several previous publications as described in the references section, and recently used in McBrian et al. (2013).


  1. Casey, J. R., Grinstein, S. and Orlowski, J. (2010). Sensors and regulators of intracellular pH. Nat Rev Mol Cell Biol 11(1): 50-61. 
  2. Kurkdjian, A. and Guern, J. (1989). Intracellular pH: measurement and importance in cell activity. Annu Rev Plant Biol 40(1): 271-303.
  3. McBrian, M. A., Behbahan, I. S., Ferrari, R., Su, T., Huang, T. W., Li, K., Hong, C. S., Christofk, H. R., Vogelauer, M., Seligson D. B. and Kurdistani, S. K. (2013). Histone acetylation regulates intracellular pH. Mol Cell 49(2): 310-321.
  4. Orij, R., Urbanus, M. L., Vizeacoumar, F. J., Giaever, G., Boone, C., Nislow, C., Brul, S. and Smits, G. J. (2012). Genome-wide analysis of intracellular pH reveals quantitative control of cell division rate by pH(c) in Saccharomyces cerevisiae. Genome Biol 13(9): R80.


细胞内pH(pH )是酶活性和细胞功能的重要生理决定因素(Kurkdjian和Guern,1989)。所有蛋白质依赖于严格调节的pH以维持其结构和功能。质子化 - 去质子化事件可以决定生物表面的电荷,并且是许多代谢反应中不可或缺的步骤(Casey等人,2010)。此外,穿过线粒体膜的质子梯度用于产生细胞能量和支持其他线粒体过程。因此,细胞已经发展了多种机制以响应于pH的胞外和细胞内波动维持pH范围的窄范围(Orij等人,2012)。在这里,我们描述了在使用荧光显微镜和pH敏感染料2',7'-双 - (2-羧乙基)-5-(和-6-氨基己酸)的活细胞中pH pH测量的方案, ) - 羧基荧光素乙酰氧基甲基酯(BCECF-AM)。该方法最近用于测定细胞内pH变化对全局组蛋白乙酰化水平的影响(McBrian等人,2013)。


  1. 赫拉细胞
  2. 尼日利亚霉素(Sigma-Aldrich,目录号:N7143) 准备在乙醇中的10mM尼日利亚菌素(1:10,000)股票(存储在-20℃的等分试样,并在实验期间保持在冰上)
  3. DMSO(Sigma-Aldrich,目录号:D2650)
  4. BCECF-AM(Life Technologies,目录号:B1170)
  5. 葡萄糖
  6. 1%抗生素 - 抗真菌剂(Gibco ,目录号:15240)
  7. DMEM(Mediatech,Cellgro ,目录号:10-013-CV)
  8. 10%FBS
  9. 石蜡
  10. 校准溶液(参见配方)
  11. 10x Earle's平衡盐溶液(EBSS),不含葡萄糖和碳酸氢钠(见配方)。
  12. 加载解决方案(参见配方)
  13. 细胞培养基(参见配方)


  1. 用于35mm盘的灌注嵌件(Warner Instruments,目录号:RC-33DM)
  2. 35mm聚赖氨酸涂布的玻璃底培养皿(MatTek,目录号:P35GC-1.0-14-C)
  3. 50ml锥形管

  4. 铝箔
  5. 配有高分辨率摄像机(ZEISS,Axio CAM MRm)的Axiovert 200 M Zeiss荧光显微镜
  6. BCECF过滤器组(Chroma Technology Corporation,目录号:71001a)
  7. 中和密度滤光片(ND 1.0A-10.0%透射率-25mm;这是针对给定显微镜定制设计的)(Chroma Technology Corporation)
  8. 管道和灌注连接
  9. 流量泵(Rainin蠕动泵Dynamax RP-1.4通道;目录号因供应商而异)
  10. 5%CO 2 罐
  11. 37°C水浴
  12. 5%CO 2培养箱(确保其被定期校准; CO 2浓度的变化可对pH产生显着影响,在实验中产生噪声)


  1. AxioVision 4.8
  2. Slidebook 4.2
  3. SigmaPlot
  4. Excel或任何电子表格分析软件


  1. 在35mm玻璃底培养皿中生长细胞(〜10 )至40%融合。 请注意,与塑料餐具相比,细胞需要较长时间才能贴附到玻璃底部菜肴。
  2. 用感兴趣的药物或siRNA处理细胞达期望的时间长度直到细胞达到〜40%融合
  3. 应该安装灌注系统以减少由于BCECF泄漏出细胞的背景信号,并且在整个pH 测量中保持培养基温度和pH恒定。
    1. 流出和流入管应具有相同的长度,以防止板的溢出或干燥。
    2. 2 ml/min是推荐的灌注速率;更快的速率可能对影响pH的细胞具有剪切效应。
    3. 为了将流出管和流入管连接到盘,我们使用针,我们弯曲成钩的形状,并用石蜡将其固定在盘上。 Premade套装可从市场购买: https://www.warneronline.com/product_info.cfm?id = 958。
    4. 为了有效的流动和防止湍流,我们使用35毫米菜肴的灌注插入和固定在与石蜡的菜。我们建议使用可见染料(如台盼蓝)测试流动的平滑度,以确保实验开始前的有效流动。
    5. 在实验前用蒸馏水冲洗灌注系统4小时,以除去管道内的任何残留物,尤其是前面实验中的尼日利亚菌素。
  4. 在37℃的水浴中设置一个架子以容纳含有与装载溶液相同的灌注溶液的50ml锥形管。类似于培养箱内的条件,在实验前平衡pH的加载溶液中以缓慢至中等速率(〜1个气泡/秒)至少20分钟的气泡5%CO 2。在实验期间继续鼓泡5%CO 2至装载溶液中以维持pH
  5. 有一个校准的pH计方便。可以连续监测装载溶液的pH以确保一致性。
  6. 在显微镜上设置灌注系统和设置。为BCECF安装色度滤波器组,并定义软件的通道。检查滤光轮,确保激发和发射通道在Axiovision软件中正确定义
  7. 确保灯泡强度为50%,过滤器密度为10%,以防止对细胞的光毒性
  8. 打开Axiovision并将曝光时间设置为100毫秒(对于440nm)和200毫秒(对于495nm)。发射设置在535nm。同时指定程序以30秒间隔拍摄6张照片。
  9. 稀释一瓶BCECF在80μlDMSO。使用预分装的BCECF粉末以防止反复冻融循环(BCECF染料在2次冻融循环后不能很好地工作)。
  10. 从细胞中取出培养基,用3ml上样溶液洗涤细胞以去除残留的培养基(某些氨基酸干扰染料加载)。
  11. 吸取2毫升1×EBSS到细胞板上,然后直接加入10微升BCECF板上。通过向上和向下移液EBSS-BCECF溶液轻轻混匀。  
  12. 将细胞在孵化器中25分钟。在孵育前5分钟开始通过灌注系统开始流动加载溶液。
  13. 为了防止光漂白,一旦从培养箱中取出并且被转移到显微镜室,就用一片铝箔包裹细胞板。关闭房间中的灯(电脑屏幕的灯应提供充足的灯光)。
  14. (在此步骤快速工作)立即将装载的菜肴安装在显微镜载物台上,并用石蜡固定在适当位置,以防止图像采集过程中的任何运动。安装灌注插入件和流入和流出管。运行灌注系统,同时聚焦显微镜找到一个合适的面积 - 约40%汇合 - 用于拍照。我们建议使用卤素灯以尽可能弱的强度进行聚焦,然后使用荧光灯进行精细聚焦,以降低细胞毒性的风险。可以使用10x-40x放大。放大10倍可以评估更多的细胞数量。
  15. 让装载溶液通过系统运行2-3分钟,然后以30秒间隔拍摄6张照片(在步骤6自动化)(图1)。

    图1. 显示了用于细胞内pH测量的最终设置。系统的组件已标记
  16. 将图片导入到Slidebook(确保在Slidebook中正确定义通道)。
  17. 导入图片后,通过选择靠近图片中间的区域(附近有一些单元格)来定义背景。然后使用遮罩工具选择单元格。在比率工具中,将495定义为分子,将450定义为分子。提取495/450通道的比率数据,并将数据导入Excel。删除异常值。使用校准方程式将比率数据转换为pH值(见下文)。

原位 校准

  1. 通过测定当在〜490nm激发染料时在535nm处的发射强度(pH依赖性)与当在〜440nm的非等静点处激发染料时的发射强度的比值来进行用BCECF的细胞内pH测量pH依赖性)。 BCECF的荧光的原位校准以相同的方式进行,但是在存在10-50μM的尼日利亚霉素和100-150mM的K on + 以平衡内部和外部pH。应对每个实验进行原位校准。
  2. 拍摄照片后,从显微镜上的装载和固定板中小心地吸取加载溶液,并使用板的侧部缓慢添加2ml校准溶液。吸出校准溶液,然后使用移液管缓慢加入2毫升校准溶液与尼日利亚菌素。等待5分钟后拍摄2张照片。在pH值5.5,6,6.5,7,7.5,8和8.5重复该过程。此时不需要流量。
  3. 将图片导入Slidebook并按上述分析,以获得每个校准pH值的495/450比例
  4. 为了将比率转换为pH,使用SigmaPlot获得最能描述数据的方程。 校准数据应符合S形曲线。 使用描述S形曲线的方程式将上面得到的实验比值转换为pH值(图2)

    图2.  典型的S形校准曲线和描述它的公式如所示


  1. 校准溶液
    135 mM KCl
    2mM K 2 HPO 2
    20 mM HEPES
    1.2mM CaCl 2 0.8mM MgSO 4 通过加入HCl或KOH将pH调节至5.5,6,6.5,7,7.5,8,8.5
  2. 10x EBSS原液(无葡萄糖和无碳酸氢钠)
    CaCl 2 2H O
    MgSO 4(无水)
    4 g/L
    NaH 2 PO 4 sub
    0.122 g/L
  3. 装载解决方案
    NaHCO 3
    24mM(pH 7.4)
    1. 所需的NaHCO 3的量由加载溶液的所需pH确定。
    2. 通过添加葡萄糖酸盐或山梨醇将溶液的摩尔渗透压浓度调整为290-310 mOsm。
    3. 准备足够的溶液,每板约25毫升。 过滤溶液并等分到50ml锥形管中。 将其盖子在5%CO 2培养箱中稍微打开以使pH平衡过夜。
  4. 细胞培养基
    Cellgro DMEM
    1%抗生素 - 抗真菌剂
    5%CO 2 2,37℃培养箱




  1. Casey,J.R.,Grinstein,S。和Orlowski,J。(2010)。 细胞内pH的传感器和调节剂。 Nat Rev Mol Cell Biol em> 11(1):50- 61.
  2. Kurkdjian,A.and Guern,J。(1989)。 细胞内pH:细胞活性的测量和重要性。 em> Annu Rev Plant Biol 40(1):271-303。
  3. McBrian,M.A.,Behbahan,I.S.,Ferrari,R.,Su,T.,Huang,T.W.,Li,K.,Hong,C.S.,Christofk,H.R.,Vogelauer,M.,Seligson D.B.and Kurdistani,S.K.(2013)。 组蛋白乙酰化调节细胞内pH 。 Mol Cell 49(2):310-321。
  4. Orij,R.,Urbanus,M.L.,Vizeacoumar,F.J.,Giaever,G.,Boone,C.,Nislow,C.,Brul,S.and Smits,G.J。(2012)。 细胞内pH的全基因组分析揭示了通过pH的细胞分裂速率的定量控制(c) em> Saccharomyces cerevisiae 。 Genome Biol 13(9):R80。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Behbahan, I. S., McBrian, M. A. and Kurdistani, S. K. (2014). A Protocol for Measurement of Intracellular pH. Bio-protocol 4(2): e1027. DOI: 10.21769/BioProtoc.1027.



amgad abdelrahman
veterinary research institute
i hope to know the protocol of measuring mineral of epithelial cell (hydrogen. phosphorus.carbon .sodium...etc)
1/13/2015 11:37:50 PM Reply
Bio-protocol team Bio-protocol team

Thank you for your suggestions. Our editorial committee will invite authors to contribute the requested protocols to our site in the near future.

1/20/2015 3:49:17 PM