Intracellular pH Measurement with Dual Excitation Fluorescence Sensor BCFL
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Intracellular pH Measurement with Dual Excitation Fluorescence Sensor BCFL

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AssayWise Letters Vol. 7(2)

Intracellular pH (pHi) plays an important modulating role in many cellular events, including cell proliferation and apoptosis, cell volume regulation, cellular metabolism, calcium regulation, receptor-mediated signal transduction, ion transport and endocytosis. Under physiological conditions, the pHi of intracellular fluid ranges between 6.8 and 7.4. This is important for many cellular functions such as protein synthesis, enzymatic activity, contractile efficiency in muscle cells and ion channel conductivity. However, neutral pH levels are not characteristic of all cellular components as some organelles require acidic environments. For example, lysosomes maintain an acidic environment (pH 4.5-6.0) to facilitate the degradation of proteins during cellular metabolism. Additionally, monitoring pHi is important for studying intracellular signaling pathways. It has been reported that the dysregulation of pHi can affect the concentration of intracellular messengers like calcium and cAMP and thus adversely influence cellular signaling.

Over the past decades various techniques have been developed for measuring pHi. These include partitioning of weak acids and bases, pH-selective microelectrodes, nuclear magnetic resonance and pH-sensitive fluorescent probes. Compared to the other pHi measurement methods, pH-sensitive probes combined with fluorescence techniques have the capacity to observe pHi changes in a spatial and temporal context. Moreover, fluorescent pH probes have high sensitivities, are non-destructive to cells and convenient to handle. A widely used fluorescent pH sensor, BCECF (2’, 7’-bis-(2-carboxyethyl)-5-(and-6) - carboxyfluorescein) was first introduced in 1982 by Roger Tsien and company (Tsien et al. 1982). It has a pKa value of 6.98 which is well within the physiological pH range of 6.8 to 7.4 making it ideal for pHi measurements. Furthermore, BCECF has the capacity to measure pH ratiometrically due to its pH-dependent dual-excitation profile. However, BCECF is not without its disadvantages. BCECF, AM (the cell permeable from) is a complex mixture of three isomers with ratios that vary between batches, and this variability can lead to complications in certain applications potentially resulting in inaccurate pHi measurements.

To overcome the isomeric variability concerning BCECF, AM, AAT Bioquest® introduces a proprietary fluorescent pH sensor, BCFL. BCFL shares an identical pKa value and spectral profiles with BCECF making it an ideal replacement for pHi applications. Ratiometric imaging with BCFL makes pHi determination significantly more accurate because variable parameters such as dye concentration, optical path length, cellular leakage and rate of photobleaching are made irrelevant. In the sections below we discuss the spectral characteristics of BCFL and experimental procedures of BCFL, AM. In two common cell lines, HeLa and HL-60, we demonstrate pHi calibration and measurement using both a flow cytometer and microplate reader. Finally, we apply BCFL in an experiment quantifying cell apoptosis based on intracellular acidification.


Spectra Characteristics of BCFL

The spectra characteristics of BCFL were measured using buffers with pH values ranging from 4.5 to 9.0. Figure 1a shows that the absorption of BCFL red shifts (450 nm to 505 nm) from pH 4.5 to 9.0 and its molar absorptivity increases to approximately 90,000 cm-1M-1. This demonstrates the pH-sensitive absorption of BCFL. Figure 1b demonstrates the emission profile of BCFL in the physiological range (pH 6-8). Similar to BCECF, BCFL has a 526 nm emission wavelength in the physiological range. Finally, Figure 1c illustrates BCFL as a dual-excitation ratiometric pH indicator with an excitation isosbestic point at 430 nm.


Figure 1. The pH dependent spectra of BCFL: a) absorbance spectra, b) emission spectra, and c) excitation spectra. The fluorescence excitation spectra on the panel c is enlarged 10x to show the isosbestic point. And the isosbestic point in excitation spectra is different from the absorption spectra.


In vitro pH Calibration and Measurement using BCFL

As a ratiometric excitation pH indicator, the measurement of pH is dependent upon the ratio of emission intensities of BCFL at two excitations, 440 nm and 505 nm (Figure 2). Ratiometric pH measurements are advantageous for its ability to correct for variations such as dye concentration, cell number, and photobleaching effects. Figure 2 illustrates that BCFL, similar to BCECF, can accurately determine pH with a standard deviation as low as 5%. Compared to the pH value of 7.33 which was measured using a pH electrode, the pH determined from the BCFL calibration curve was 7.30, demonstrating a 2% error from 8 replicates. Additionally, the calibration curve shows BCFL has a pKa value of ~6.8 which is comparable to the pKa of BCECF.


Figure 2. The pH dependent standard curve of BCFL using buffers with varying pH values. An average of 3 data points was plotted and a sigmoidal trendline was fitted to get the pH standard curve. +/- error bars represent the standard deviation.


Intracellular pH Measurement by Plate Reader

The non-fluorescent acetoxymethyl ester of BCFL (BCFL, AM) is cell membrane-permeant probe that can be applied to in situ experiments measuring pHi. After BCFL, AM noninvasively enters cells, AM ester functional groups are removed by intracellular esterase hydrolysis. This improves the cellular retention of BCFL by restoring its negative charge and simultaneously activates the pH-sensitive fluorescent properties of BCFL. BCFL, AM’s pHi calibration curve is sigmoidal from 4.5 to 9.0 with its linear range falling in the cytosolic physiological pH range of 6 - 8 (Figure 3). The pKa from this calibration curve is ~6.8 which is almost identical to the in vitro calibration curve illustrated in Figure 2.


Figure 3. Standard curve created using BCFL, AM (Cat# 21190) with Intracellular pH Calibration Buffer Kit. Hela cells were incubated with 5 uM BCFL, AM for 30 minutes 37 oC. The Intracellular pH Calibration Buffer Kit (Cat#21135) was used to clamp the intracellular pH with extracellular buffers at pH 4.5 to 9.0. An average of 4 data points was plotted and a sigmoidal trendline line was fitted to get the pH standard curve from 5 to 8.


Figure 4. Hela cells labeled with BCFL, AM Intracellular pH Indicator. Hela cells were incubated with 5 µM of BCFL, AM Intracellular pH Indicator (Cat#21190) for 30 minutes at 37 oC. Images were captured using a microscope FITC filter. Incubation of BCFL, AM solution with Hela cells showed a homogenous uptake of BCECF (Fig. 6) and stained cell cytosol.


Intracellular pH Measurement by Flow Cytometry:

Intracellular pH measurement by flow cytometry is a generally procedure that can be done using any instrument equipped with a 488-nm argon laser. But ratio measurement is not easily performed with flow cytometers because of the limitation of the excitation laser.  Although there is no universal calibration curve for internal pH calibration, the pH change could be easily straightforwardly determined with flow cytometers.


Figure 5.  HL-60 cells incubated with BCFL, AM (Cat#21190) and clamped with intracellular pH calibration buffer (pH=4.5 & pH=8.5). HL-60 cells were incubated with 100nM BCFL, AM for 30 minutes at 37 oC. The Intracellular pH Calibration Buffer Kit (Cat. 21235) was used to clamp the intracellular pH with extracellular buffer at pH 4.5 (Green area) and pH 8.5 (Red area). Unstained cells are represented by the Blue area. Samples were acquired and analyzed using Acea Novocyte Cytometer.


Application of BCFL in Cell Apoptosis:

Under normal conditions, pHi is actively maintained within a very narrow pH range of 6.0 – 8.0. However, during certain important cellular processes, such as cell proliferation or apoptosis, pHi values change and therefore serve as a cell signaling factor. During apoptosis, cells induce intracellular acidification to pH values which activate enzyme reactions necessary for programmed cell death.

Intracellular acidification was observed during apoptosis in Jurkat cells after treatment with staurosporine for 4 hours. In Figure 6 staurosporine treated Jurkat cells show lower fluorescence intensities in the FITC channel, indicating the intracellular acidification of Jurkat cells during apoptosis. Acidification was also noted in the population of smaller size cells. In the earlier stage of apoptosis, the pHi shows no significant change, but as the process of apoptosis progresses, the cells decrease in size and become more acidic (Figure 6). This decrease in pHi can serve as a practical indicator of cellular deterioration and cell death.


Figure 6. BCFL, AM & AnnexinV-iF647 Staining of Apoptosis Jurkat cells. Junket cells were treated with/without 1uM staurosporine in growth medium for ~4 hours. After treatment, cells were stained with BCFL, AM & AnnexinV-iF647 and DAPI cocktail in HH Buffer for 30min, and dye loading solution was then replaced with HH buffer and analyzed with ACEA NovoCyte Flow Cytometer with DAPI, FITC and APC channels. 


Conclusion

Overall, we demonstrate BCFL is an ideal dual excitation fluorescence pH sensor for quantifying pHi and a suitable replacement for BCECF. We demonstrated that with BCFL, the physiological pHi can be determined from calibration curves derived from flow cytometer and microplate reader formats. Most importantly, the single isomer of BCFL, AM makes pH measurements much more reproducible than BCECF, AM which suffers from isomeric variability.


Table 1. Product Ordering Information

Cat. #Product NameUnit SizePrice
21190
RatioWorks™ BCFL, AM1 mg$145
21191
RatioWorks™ BCFL, SE1 mg
$195
21189
RatioWorks™ BCFL, Acid1 mg$95


MATERIALS AND METHODS

Materials:

BCFL (Cat# 21189) and BCFL, AM (21190) were used for in vitro pH calibration and intracellular pH measurement, and BCECF (Cat#21201) and BCECF, AM (Cat#21202) were also used as a reference. Internal pH calibration buffers were from Spexyte™ Intracellular pH Calibration Buffer Kit (Cat# 21235). The 4-parameter curve pH calibration curves were plotted with AAT Bioquest's Four Parameter Logistic (4PL) Curve Calculator.

Cell Culture:

HeLa cells were cultured in DMEM medium with 10% FBS and 1% Penicillin-Streptomycin-Glutamine. HL-60 and Jurkat cells were cultured in RPMI medium with 10% FBS and 1% Penicillin-Streptomycin-Glutamine. When indicated, the cells were treated with 1 µM staurosporine in growth cells directly, and followed by culturing for ~4 hours to induce cell apoptosis.

Instruments:

The fluorescence intensities was measured by FlexStation 3 (Molecular Devices) bottom reading mode with Ex/Em=505 nm, 440 nm/535 nm (cutoff=515 nm). Flow cytometry data were acquired and analyzed with NovoCyte Flow Cytometry (ACEA) using FITC channel. All cell images were captured with a BZ-X710 fluorescence microscope using FITC filter set.

Intracellular pH Calibration and Measurement with 96-well plate platform:

Sample Protocol for intracellular pH nigericin calibration

  1. HeLa Cells were plated in 100 µL culture medium in 96-well clear bottom black plates at 50,000 cells per well.
  2. Medium was removed and 100 µL HH buffer with 0.04% PF127 with 5 µM BCFL, AM was added to each well and cells were incubated at 37 oC for 30 min to 60 min.
  3. The dye loading solution was removed.
  4. 100 µL Intercellular pH Calibration (Cat# 21235) with 10 µM Nigerincin & 10 µM Valinomycin were added to each well and then incubated at room temperature for 10-30 min.
  5. The fluorescence was measured by FlexStation 3 (Molecular Devices) bottom reading mode with Ex/Em=505 nm, 440 nm/530 nm (cutoff=515 nm).

Intracellular pH Calibration and Measurement with Flow Cytometry Platform:

Sample Flow Cytometry Protocol for Intercellular pH calibration:

  1. HL-60 cells were grown in culture medium overnight
  2. The next day, 5 mL cells were prepared in a tube
  3. Centrifuge to remove growth medium
  4. Add 1 mL HH buffer with 0.02% PF127 and 100 nM BCFL,AM
  5. Incubate for 30 min at 37 oC
  6. Centrifuge to remove dye loading solution
  7. Resuspend cells in 1 mL HH buffer
  8. Prepare 1.0 mL of each intracellular pH buffer with 10 µM Nigerincin & 10 µM Valinomycin
  9. Add 100 µL cells to each intracellular pH calibration buffer
  10. Incubate for 10-30 min at room temperature, and then analyze with a flow cytometer using FITC channel


References

  1. Rink, T. J. “Cytoplasmic PH and Free Mg2 in Lymphocytes.” The Journal of Cell Biology, vol. 95, no. 1, Jan. 1982, pp. 189–196., doi:10.1083/jcb.95.1.189.
  2. Zhu, Wen-Hui, and Tatt-Tuck Loh. "Effects of Na+/H+ antiport and intracellular pH in the regulation of HL-60 cell apoptosis." Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1269.2 (1995): 122-128.
  3. Gottlieb, Roberta A., et al. "Apoptosis induced in Jurkat cells by several agents is preceded by intracellular acidification." Proceedings of the National Academy of Sciences 93.2 (1996): 654-658.
  4. Shrode, Lamara D., Hans Tapper, and Sergio Grinstein. "Role of intracellular pH in proliferation, transformation, and apoptosis." Journal of bioenergetics and biomembranes 29.4 (1997): 393-399.
  5. Thomas, John A., et al. "Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ." Biochemistry 18.11 (1979): 2210-2218.
  6. Valet, G., et al. "Fast intracellular pH determination in single cells by flow-cytometry." Naturwissenschaften 68.5 (1981): 265-266.
  7. Arosio, Daniele, et al. "Simultaneous intracellular chloride and pH measurements using a GFP-based sensor." Nature methods 7.7 (2010): 516.
  8. Casey, Joseph R., Sergio Grinstein, and John Orlowski. "Sensors and regulators of intracellular pH." Nature reviews Molecular cell biology 11.1 (2010): 50.