Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Summary

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.

Abstract

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 pinocytosis^3,4. 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.

Protocol

1. Preparation of Solutions for Cellular pH Calibration

1. Add the following compounds to five separate 50 ml conical tubes to prepare 5 solutions to be used for calibration:
   • NaCl (1 ml of 1 M NaCl) (1 M NaCl = 0.58 g in 10 ml H₂0)
   • KCl (6.75 ml of 1 M KCl) (1 M KCl = 0.75 g in 10 ml H₂0)
   • Glucose (1 ml of 1 M D-Glucose) ( 1M D-Glucose = 1.80 g in 10 ml H₂0)
   • MgS0₄ (0.05 ml of 1 M MgSO₄) (1 M MgSO₄= 1.20 g in 10 ml H₂0)
   • 20 mM HEPES (1 ml of 1M HEPES) (1 M HEPES = 2.38 g in 10 ml H₂0)
   • CaCl

Results

For the simultaneous measurement of pH in both intracellular and endosomal/lysosomal cell compartments, the ratiometric fluorescent pH-sensing probes SNARF-1 and HPTS were used. SNARF-1 is restricted to the cytosolic compartment whereas HPTS allows ratiometric pH measurement of the endosomal/lysosomal compartment. Cell loading with HPTS for 16 hr allows its selective localization in endosomes/lysosomes⁴. A typical example of fluorescence recorded from HT-1080 cells loaded with the pH-sensing probes is shown in.......

Discussion

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 microinjection^8-10. These techniques suffer from serious drawbacks, namely electroporation caus.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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