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 Sant&#233; (FRSQ)-funded Centre de Recherche Clinique &#201;tienne LeBel.

1. Preparation of Solutions for Cellular pH Calibration
<ol>
	<li>Add the following compounds to five separate 50 ml conical tubes to prepare 5 solutions to be used for calibration:
	<ul>
		<li>NaCl (1 ml of 1 M NaCl) (1 M NaCl = 0.58 g in 10 ml H20)</li>
		<li>KCl (6.75 ml of 1 M KCl) (1 M KCl = 0.75 g in 10 ml H20)</li>
		<li>Glucose (1 ml of 1 M D-Glucose) ( 1M D-Glucose = 1.80 g in 10 ml H20)</li>
		<li>MgS04 (0.05 ml of 1 M MgSO4) (1 M MgSO<s...

<ol>
	<li>Demaurex, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pH+Homeostasis+of+cellular+organelles.">pH Homeostasis of cellular organelles.</a> News Physiol Sci. 17, 1-5 (2002).</li><li>Webb, B. A., Chimenti, M., Jacobson, M. P., Barber, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulated+pH:+a+perfect+storm+for+cancer+progression.">Dysregulated pH: a perfect storm for cancer progression.</a> Nat Rev Cancer. 11, 671-677 (2011).</li><li>Hinton, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+a+subunit+isoforms+of+the+V-ATPase+in+pH+homeostasis+and+in+vitro+invasion+of+MDA-MB231+human+breast+cancer+cells.">Function of a subunit isoforms of the V-ATPase in pH homeostasis and in vitro invasion of MDA-MB231 human breast cancer cells.</a> J Biol Chem. 284, 16400-16408 (2009).</li><li>Overly, C. C., Lee, K. D., Berthiaume, E., Hollenbeck, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+intraorganelle+pH+in+the+endosomal-lysosomal+pathway+in+neurons+by+using+ratiometric+imaging+with+pyranine.">Quantitative measurement of intraorganelle pH in the endosomal-lysosomal pathway in neurons by using ratiometric imaging with pyranine.</a> Proc Natl Acad Sci U S A. 92, 3156-3160 (1995).</li><li>Drose, S., Altendorf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bafilomycins+and+concanamycins+as+inhibitors+of+V-ATPases+and+P-ATPases.">Bafilomycins and concanamycins as inhibitors of V-ATPases and P-ATPases.</a> J Exp Biol. 200, 1-8 (1997).</li><li>Pedersen, S. F., King, S. A., Nygaard, E. B., Rigor, R. R., Cala, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NHE1+inhibition+by+amiloride-+and+benzoylguanidine-type+compounds.+Inhibitor+binding+loci+deduced+from+chimeras+of+NHE1+homologues+with+endogenous+differences+in+inhibitor+sensitivity.">NHE1 inhibition by amiloride- and benzoylguanidine-type compounds. Inhibitor binding loci deduced from chimeras of NHE1 homologues with endogenous differences in inhibitor sensitivity.</a> J Biol Chem. 282, 19716-19727 (2007).</li><li>Alfonso, A., Cabado, A. G., Vieytes, M. R., Botana, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-pH+crosstalks+in+rat+mast+cells:+cytosolic+alkalinization,+but+not+intracellular+calcium+release,+is+a+sufficient+signal+for+degranulation.">Calcium-pH crosstalks in rat mast cells: cytosolic alkalinization, but not intracellular calcium release, is a sufficient signal for degranulation.</a> Br J Pharmacol. 130, 1809-1816 (2000).</li><li>Han, J., Burgess, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+indicators+for+intracellular+pH.">Fluorescent indicators for intracellular pH.</a> Chem Rev. 110, 2709-2728 (2010).</li><li>Giuliano, K. A., Gillies, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+intracellular+pH+of+BALB/c-3T3+cells+using+the+fluorescence+of+pyranine.">Determination of intracellular pH of BALB/c-3T3 cells using the fluorescence of pyranine.</a> Anal Biochem. 167, 362-371 (1987).</li><li>Willoughby, D., Thomas, R., Schwiening, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+intracellular+pH+changes+on+resting+cytosolic+calcium+in+voltage-clamped+snail+neurones.">The effects of intracellular pH changes on resting cytosolic calcium in voltage-clamped snail neurones.</a> J Physiol. 530, 405-416 (2001).</li></ol>

No conflict of interest declared.

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...

<table border="0" cellpadding="0" cellspacing="0" width="872">
	<tbody>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">Solutions for calibration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Sodium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">L-11621</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Potassium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M-12445</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">D-Glucose (dextrose)</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">D16-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Magnesium sulfate</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M65-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">HEPES</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Calcium chloride dihydrate</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M-1612</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Deionized water</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Nigericin</td>
			<td style="width:136px;">Calbiochem</td>
			<td align="right" style="width:119px;">481990</td>
			<td>Toxic by inhalation or in contact with skin</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td style="width:315px;">Keep protected from the light at 4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Culture dishes &#8709; 35mm</td>
			<td style="width:136px;">BD Biosciences</td>
			<td align="right" style="width:119px;">354456</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">pH-sensing probes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS)</td>
			<td style="width:136px;">Life technologies</td>
			<td style="width:119px;">H-348</td>
			<td>Keep away from the light at room temperature</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">5-(and-6)-Carboxy SNARF&#174;-1, Acetoxymethyl Ester, Acetate</td>
			<td style="width:136px;">Life technologies</td>
			<td style="width:119px;">C-1271</td>
			<td>Keep away from the light and humidity at -20&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Dimethyl Sufoxide (DMSO)</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">BP231-1&#160;</td>
			<td>Harmful</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">Phosphate buffered saline (PBS) 100X</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Potassium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M-12445</td>
			<td>20 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Sodium phosphate monobasic</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">S369-1</td>
			<td>115 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Potassium phosphate monobasic</td>
			<td style="width:136px;">J.T Baker</td>
			<td style="width:119px;">3246-01</td>
			<td>20 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Deionized water</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>Bring to 1 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Autoclave and adjust pH to 7.4</td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">Phosphate buffered saline (PBS) 1X</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">PBS 100X</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>50 mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Sodium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">L-11621</td>
			<td>40,5 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Deoinized water</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>Bring to 5 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">pH modulators</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">&#160;Bafilomycin A1</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:119px;">B-1793</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-(N-Ethyl-N-isopropyl)amiloride (EIPA)</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:119px;">A3085</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Ammonium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">A649-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

simultaneous ph measurement in endocytic and cytosolic compartments in living cells using confocal microscopy

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.

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...

Watch this Scientific Journal Video about Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy at JoVE.com

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

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 ...

simultaneous-ph-measurement-endocytic-cytosolic-compartments-living

Research

JoVE Journal

Biology

14.9K visualizações.  Université de Sherbrooke. 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. 