Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.
Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases2. Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells3. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.
Quantitative pH measurement in live-cell imaging of different cellular compartments is needed to accurately measure pH variations in cell responses to external challenges. However, one of the major hurdles that remains is to easily and specifically target cellular compartments. In this respect, a few studies have highlighted the use of HPTS as an intracellular pH indicator introduced into cells by electroporation or microinjection8-10. These techniques suffer from serious drawbacks, namely electroporation caus…
The authors have nothing to disclose.
We thank Dr. Gilles Dupuis for critical reading of the manuscript.
Funding
This work was supported by the Canadian Institutes for Health Research, grant # MOP-126173. C.M.D is a member of the Fonds de la Recherche en Santé (FRSQ)-funded Centre de Recherche Clinique Étienne LeBel.
Solutions for calibration | |||
Sodium chloride | Fischer | L-11621 | |
Potassium chloride | Fischer | M-12445 | |
D-Glucose (dextrose) | Fischer | D16-1 | |
Magnesium sulfate | Fischer | M65-500 | |
HEPES | Sigma-Aldrich | H3375 | |
Calcium chloride dihydrate | Fischer | M-1612 | |
Deionized water | N/A | N/A | |
Nigericin | Calbiochem | 481990 | Toxic by inhalation or in contact with skin |
Keep protected from the light at 4°C | |||
Culture dishes ∅ 35mm | BD Biosciences | 354456 | |
pH-sensing probes | |||
8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS) | Life technologies | H-348 | Keep away from the light at room temperature |
5-(and-6)-Carboxy SNARF®-1, Acetoxymethyl Ester, Acetate | Life technologies | C-1271 | Keep away from the light and humidity at -20°C |
Dimethyl Sufoxide (DMSO) | Fischer | BP231-1 | Harmful |
Phosphate buffered saline (PBS) 100X | |||
Potassium chloride | Fischer | M-12445 | 20 g |
Sodium phosphate monobasic | Fischer | S369-1 | 115 g |
Potassium phosphate monobasic | J.T Baker | 3246-01 | 20 g |
Deionized water | N/A | N/A | Bring to 1 L |
Autoclave and adjust pH to 7.4 | |||
Phosphate buffered saline (PBS) 1X | |||
PBS 100X | N/A | N/A | 50 mL |
Sodium chloride | Fischer | L-11621 | 40,5 g |
Deoinized water | N/A | N/A | Bring to 5 L |
pH modulators | |||
Bafilomycin A1 | Sigma-Aldrich | B-1793 | |
5-(N-Ethyl-N-isopropyl)amiloride (EIPA) | Sigma-Aldrich | A3085 | |
Ammonium chloride | Fischer | A649-500 |