Konfokal mikroskopi kullanılarak Yaşayan hücreler içinde endositik ve Sitosolik Bölümleri eşzamanlı pH Ölçümü
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 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.
Protocol
Representative Results
Discussion
Farklı hücre bölümlerinde canlı hücre görüntüleme Sayısal pH ölçümü doğru dış zorluklara hücre yanıtlarında pH değişimleri ölçmek için gereklidir. Bununla birlikte, hala önemli engellerden biri kolayca ve özel hücre bölmeleri hedef etmektir. Bu bağlamda, bir kaç çalışmada, elektroporasyon ya da mikroenjeksiyon ile 8-10 hücrelere katılan bir hücre içi pH göstergesi olarak HPTS kullanımını sermiştir. Bu teknikler yani elektro hücrelere geniş zararlara yol açar, ve m…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 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 | |
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