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. Preparazione di soluzioni per cellulare pH Calibrazione <ol><li> Aggiungere i seguenti composti da cinque tubi 50 ml coniche separati per preparare 5 soluzioni da utilizzare per la calibrazione: <ul><li> NaCl (1 ml di 1 M NaCl) (1 M NaCl = 0,58 g in 10 ml di H 2 0) </li><li> KCl (6,75 ml di 1 M KCl) (1 M KCl = 0,75 g in 10 ml di H 2 0) </li><li> Glucosio (1 ml di 1 M D-glucosio) (1M D-Glucosio = 1,80 g in 10 ml di H 2 0) </li><li> MgS0 4 (0,05 ml di 1 M MgSO 4) (1 M MgSO 4 = 1,20 g in 10 ml di H 2 0) </li><li> HEPES 20 mM (1 ml di 1M HEPES) (1 M HEPES = 2,38 g in 10 ml di H 2 0) </li><li> CaCl 2 (0,05 ml di 1M CaCl 2) (1 M CaCl 2 = 1,47 g in 10 ml di H 2 0) Preparare tutte le soluzioni a doppia distillata H 2 O (DDH 2 O). </li></ul></li><li> Completare ogni soluzione con 35 ml di DDH 2 O e mescolare con un agitatore magnetico fino a tantoluzione è raggiunto. </li><li> Regolare il pH di ciascuna soluzione con 1 N KOH o 1 N HCl per ottenere valori di pH 5.5, 6.0, 6.5, 7.0 e 7.5. Poi, regolare il volume con acqua deionizzata fino ad un volume finale di 50 ml. </li><li> Misura 5 mg di nigericin e aggiungerlo a 670 ml di EtOH assoluto per preparare un 10 mM nigericin soluzione stock. Attenzione: nigericin è altamente tossico e deve essere maneggiata con molta cura (guanti, occhiali di protezione e maschera). Miscelare la soluzione per inversione fino a quando il nigericin è completamente sciolto. </li><li> Aggiungere 0,05 ml di nigericin 10 mm (concentrazione finale 10 mM) ad ogni soluzione di taratura. Nigericina è uno ionoforo che aumenta la permeabilità della membrana cellulare e provoca equilibrazione di pH intracellulare ed extracellulare in presenza di una concentrazione depolarizzante di K + extracellulare. </li><li> Soluzioni di taratura possono essere memorizzati fino a 1 mese a 4 ° C </li></ol> 2. Preparazione cellulare <ol><li> Sotto un safet biologicoCappa y, cellule seme in 35 millimetri x 10 millimetri piatti di coltura in 2 ml di terreno di coltura supplementato con 10% di siero fetale bovino e antibiotici. Usare la quantità di cellule necessarie per garantire circa il 50% di confluenza dopo 24 ore. NOTA: Nei nostri esperimenti, usiamo la linea cellulare HT-1080 e la piastra 1 x 10 5 cellule per capsula di Petri. </li><li> Determinare il numero di piatti di coltura necessari per l&#39;esperimento e piastra 5 ulteriori piastre di coltura da utilizzare per la calibrazione. </li><li> Piatti di porre la cultura in un C 5% CO 2 incubatore 37 ° / per almeno 24 ore. </li></ol> 3. Preparazione di sonde pH-sensing <ol><li> Preparare una soluzione madre 1M della sonda pH fluorescente 8-idrossipirene-1, 3,6 acido trisolfonico, sale trisodico (HPTS / pyranine) in DDH 2 0. Questa soluzione deve essere conservato a temperatura ambiente e al riparo dalla luce. </li><li> Preparare una soluzione stock di 1 mM 5 - (e-6)-carbossilico seminaphthorhodafluor acetossimetilico estere acetato (C-SNARF-01:00; chiamato thereaopo SNARF-1) sciogliendo 1 mg di SNARF-1 in 1,76 ml di DMSO. Mescolare accuratamente pipettando ripetuta. SNARF-1 soluzione deve essere conservata a -20 ° C e al riparo dalla luce. </li></ol> 4. Etichettatura cellulare <ol><li> Sedici ore prima del live-cell imaging, aggiungere 2 ml di soluzione stock 1M HPTS (concentrazione finale 1 mm) per ogni piatto di coltura contenente 2 ml di terreno completo. </li><li> Riportare i piatti della cultura per l&#39;incubatore. La sonda HPTS è cell-impermeant, quindi etichetta organelli la sonda deve essere occupato da pinocitosi. Questa condizione richiede che incubazione delle cellule con HPTS vengono eseguite in medium siero supplementato cultura. </li><li> Dopo l&#39;incubazione con HPTS, lavare le cellule due volte con PBS sterile e aggiungere 2 ml di mezzo privo di siero per almeno 2 ore. Incubazione con SNARF-1 deve essere eseguita in terreno privo di siero perché siero può impedire l&#39;assorbimento della sonda. </li><li> Aggiungere 10 ml di un SNARF-1 soluzione stock 1 mm per ottenere afconcentrazione inale su 5 micron e consentono alle cellule di incubare per 20 min a 37 ° C, sotto la luce fioca. C-1-SNARF concentrazione e tempo di incubazione può variare a seconda del tipo cellulare. </li><li> Al termine dell&#39;incubazione, lavare le cellule due volte in PBS sterile e aggiungere 2 ml di mezzo privo di siero. </li></ol> 5. Aggiunta di pH modulatori <ol><li> Per valutare se il metodo può rilevare variazioni di pH nelle cellule, diversi reagenti che modulano pH intracellulare possono essere utilizzati: <ul><li> Bafilomycin A1, un inibitore specifico della V-ATPasi 5 </li><li> Na + / H + scambiatore isoforma 1 (NHE-1) 6 </li><li> NH 4 Cl, una base debole che aumenta intracellulare pH 7 </li></ul><ol><li> Preparare soluzioni madre dei reattivi di cui sopra: <ul><li> Bafilomycin A1: aggiungere 10 mg di bafilomycin A1 a 160 ml di DMSO per ottenere una soluzione stock di 100 micron </li><li> EIPA: aggiungere 25 mg di EIPA a 1.67 ml di metanolo per ottenuna concentrazione finale di 50 mM </li><li> NH 4 Cl: sciogliere 0,53 g di cloruro di ammonio in 10 ml di DDH 2 O per ottenere una concentrazione finale di 1 mM. </li></ul> Nota: Tutte le soluzioni di archivi devono essere conservati a 4 ° C ad eccezione di bafilomycin A1, che deve essere conservato a -20 ° C e al riparo dalla luce. </li><li> In piatti della cultura separati, aggiungere uno dei reagenti come descritto di seguito: <ul><li> 2 ml di 100 mM bafilomycin A1 (concentrazione finale 100 nm) </li><li> 1 ml di 50 mM EIPA (concentrazione finale 25 pm) </li><li> 40 ml di 1M NH 4 Cl (concentrazione finale 20 mM) </li></ul></li><li> Incubare le cellule per 30 min a 37 ° C. </li></ol></li><li> Procedere alla acquisizione delle immagini. </li></ol> 6. Specifiche tecniche di imaging microscopio live-cell e impostazioni di acquisizione <ol><li> Eseguire live-cell imaging usando un obiettivo 40X montato su una spettrale invertito confocale a scansione microscope dotato di un palco XY motorizzato, una camera che ospita fondo di vetro piatti della cultura in un (temperatura, umidità, CO 2) fase di incubatore ambiente controllato. </li><li> Lo strumento confocale (Olympus FV1000) qui usato è dotato di: <ul><li> Un diodo laser 405 nm </li><li> Un laser ad argon 40 mW (458 nm, 488 nm, 515 nm) </li><li> Un laser elio neon (543 nm) </li><li> Obiettivo Fluorite 10X, NA0.4 </li><li> Un obiettivo LUCPLFLN 40X, NA0.60 </li><li> Un obiettivo olio PLAPONSC PIANO APO 60X, NA1.4 </li><li> Uno specchio dicroico DM 405/458/488/543 </li><li> Un SDM 560 e un SDM 640 filtri dicroici </li><li> Un filtro barriera BA655-755 </li><li> Un BS20/80 divisore di fascio </li><li> Software di analisi dei dati per acquisizione ed elaborazione, </li></ul></li><li> Una volta che l&#39;incubatore fase si è riscaldata fino a 37 ° C e il microscopio è istituito, mettere un piatto di coltura precedentemente incubate con HPTS e SNARF-1 nella camera di imaging fase confocale. </li><li> Laopo l&#39;individuazione di una zona di interesse, regolare le impostazioni di acquisizione del software Figura 1. <ul><li> Impostare due set di canali virtuali. </li><li> Avviare la prima fase che corrisponde al HPTS eccitazione a 405 nm e la raccolta emissione a 505-525 nm e SNARF-1 eccitazione a 543 nm e la raccolta emissione a 570-600 nm. </li><li> Avviare la seconda fase che corrisponde alla HPTS eccitazione a 458 nm e la raccolta emissione a 505-525 nm e SNARF-1 eccitazione a 543 nm e la raccolta emissione a 640-650 nm. </li><li> L&#39;apertura confocale è impostato a 175 micron per default e apertura ottimale deve essere impostata manualmente a 300 micron. </li><li> Prendere Immagine impilando seriali sezioni ottiche orizzontali (800 x 800 pixel) da 0,4 micron di spessore. </li></ul></li></ol> NOTA: Un obiettivo 40X è utile osservare più celle contemporaneamente e funziona bene per la visualizzazione organello. Tuttavia, la risoluzione spaziale e l&#39;intensità del segnale sono better ottenuto utilizzando un obiettivo 60X olio. Inoltre, non c&#39;è eccitazione o di emissione di diafonia tra i due coloranti. 7. Taratura delle sonde di pH-sensing <ol><li> Prima della taratura, rimuovere terreno dalla piastra di coltura precedentemente incubate con HPTS e SNARF-1, lavare due volte con PBS sterile e poi aggiungere 2 ml di una delle cinque soluzioni di taratura. </li><li> Posizionare il piatto cultura nella camera di fase confocale. </li><li> Procedere al time-lapse acquisizione delle immagini secondo le seguenti impostazioni: <ol><li> Impostare il numero di immagini e tempi di durata degli intervalli per l&#39;esperimento. NOTA: Usiamo tipicamente un intervallo di 1 minuto tra le immagini fino a 30 min. </li><li> Programmare l&#39;otturatore per chiudere tra l&#39;acquisizione di ogni immagine. </li></ol></li><li> Registrare l&#39;intensità di fluorescenza per min in un foglio elettronico e determinare il momento in cui l&#39;intensità di fluorescenza è costante. PH intracellulare è generalmente stabile dopo 15 - 20 min. </li><li> Ripetere passaggi 7,1-7,4 utilizzando un nuovo piatto di coltura per ogni soluzione di calibrazione rimanente. </li></ol> 8. Acquisizione dati <ol><li> Una volta che la calibrazione è stato fatto, acquisire dati di esempio 4-5 campi scelti a caso contengono 15-20 cellule per campo. Due immagini sono ottenute per ciascun campo: uno per l&#39;etichettatura HPTS (vescicole) e il secondo per SNARF-1 etichettatura (citosol). </li><li> Fluorescenza di fondo (zona extracellulare) viene digitalmente sottratto dalle immagini fluorescenti di entrambe le sonde. Vescicole etichettati e citoplasma dei rispettivi immagini memorizzate sono state selezionate usando una maschera binaria. Per ogni sonda pH-sensing, intensità di fluorescenza sono misurati su una scala numerica (0-4,095 scala di livelli di grigio). Una intensità medio per tutti i pixel del organello o citoplasma è calcolato ei valori vengono utilizzati per calcolare i rapporti di fluorescenza. </li></ol> 9. Analisi dei dati <ol><li> I vari pacchetti software sono disponibili in commercio per l&#39;analisi dei dati. EsempioFluoView Software. </li><li> In un foglio di calcolo, i valori dei record e calcolare rapporti di fluorescenza come segue: Nel caso di HPTS, R = (F 458 nm / F 405 nm) e, nel caso di SNARF-1, R = (F 644 nm / F 584 nm ). </li><li> Tracciare i dati utilizzando software grafici scientifici come GraphPad Prism. La trama dovrebbe ritrarre rapporti di fluorescenza (asse y) in funzione del pH (asse X). Curva di raccordo è realizzato utilizzando la regressione non lineare. </li><li> Raccogliere intensità di fluorescenza dei campioni come descritto al punto 8.2 e calcolare rapporti di fluorescenza. Usare curve di calibrazione per trasformare i rapporti in valori di pH. </li></ol>

<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.

Misura di pH quantitativa in live-cell imaging di diversi compartimenti cellulari è necessaria per misurare con precisione le variazioni di pH nelle risposte cellulari alle sfide esterne. Tuttavia, uno dei principali ostacoli che rimane è facile e specificamente i compartimenti cellulari. A questo proposito, alcuni studi hanno evidenziato l&#39;uso di HPTS come indicatore di pH intracellulare introdotto in cellule mediante elettroporazione o microiniezione 8-10. Queste tecniche soffrono di gravi inconvenien...

<table><tbody><tr><td>Solutions for calibration</td><td></td><td></td><td></td></tr><tr><td>Sodium chloride</td><td>Fischer</td><td>L-11621</td><td></td></tr><tr><td>Potassium chloride</td><td>Fischer</td><td>M-12445</td><td></td></tr><tr><td>D-Glucose (dextrose)</td><td>Fischer</td><td>D16-1</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Fischer</td><td>M65-500</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Fischer</td><td>M-1612</td><td></td></tr><tr><td>Deionized water</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Nigericin</td><td>Calbiochem</td><td>481990</td><td>Toxic by inhalation or in contact with skin</td></tr><tr><td colspan="4">Keep protected from the light at 4 &amp;deg;C</td></tr><tr><td>Culture dishes &amp;empty; 35mm</td><td>BD Biosciences</td><td>354456</td><td></td></tr><tr><td>pH-sensing probes</td><td></td><td></td><td></td></tr><tr><td>8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS)</td><td>Life technologies</td><td>H-348</td><td>Keep away from the light at room temperature</td></tr><tr><td>5-(and-6)-Carboxy SNARF&amp;reg;-1, Acetoxymethyl Ester, Acetate</td><td>Life technologies</td><td>C-1271</td><td>Keep away from the light and humidity at -20 &amp;deg;C</td></tr><tr><td>Dimethyl Sufoxide (DMSO)</td><td>Fischer</td><td>BP231-1&amp;nbsp;</td><td>Harmful</td></tr><tr><td>Phosphate buffered saline (PBS) 100X</td><td></td><td></td><td></td></tr><tr><td>Potassium chloride</td><td>Fischer</td><td>M-12445</td><td>20 g</td></tr><tr><td>Sodium phosphate monobasic</td><td>Fischer</td><td>S369-1</td><td>115 g</td></tr><tr><td>Potassium phosphate monobasic</td><td>J.T Baker</td><td>3246-01</td><td>20 g</td></tr><tr><td>Deionized water</td><td>N/A</td><td>N/A</td><td>Bring to 1 L</td></tr><tr><td>Autoclave and adjust pH to 7.4</td><td></td><td></td><td></td></tr><tr><td>Phosphate buffered saline (PBS) 1X</td><td></td><td></td><td></td></tr><tr><td>PBS 100X</td><td>N/A</td><td>N/A</td><td>50 ml</td></tr><tr><td>Sodium chloride</td><td>Fischer</td><td>L-11621</td><td>40.5 g</td></tr><tr><td>Deoinized water</td><td>N/A</td><td>N/A</td><td>Bring to 5 L</td></tr><tr><td>pH modulators</td><td></td><td></td><td></td></tr><tr><td>Bafilomycin A1</td><td>Sigma-Aldrich</td><td>B-1793</td><td></td></tr><tr><td>5-(N-Ethyl-N-isopropyl)amiloride (EIPA)</td><td>Sigma-Aldrich</td><td>A3085</td><td></td></tr><tr><td>Ammonium chloride</td><td>Fischer</td><td>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.

Per la misura simultanea di pH sia intracellulare e compartimenti cellulari endosomal / lisosomiali, sono stati usati i fluorescenti sonde pH-sensing raziometrici Snarf-1 e HPTS. SNARF-1 è limitata al compartimento citosolico che HPTS permette di misurare raziometrica pH del vano endosomale / lisosomiale. Carico delle cellule con HPTS per 16 ore permette la sua localizzazione selettiva negli endosomi / lisosomi 4. Un tipico esempio di fluorescenza registrata da cellule HT-1080 caricato con le sonde pH-sensib...

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.

La misurazione simultanea di pH in endocitosi e citosoliche scomparti nelle cellule viventi usando Microscopia confocale

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

14.9K Views.  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. 

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

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used for ATP production. The amphiphilic nature of membrane proteins requires particular attention to their handling, and reconstitution into the natural lipid environment is indispensable when studying membrane transport processes like proton translocation. Here, we detail a method that has been used for the investigation of the proton-pumping mechanism of membrane redox enzymes, taking cytochrome bo3 from Escherichia coli as an example. A combination of electrochemistry and fluorescence microscopy is used to control the redox state of the quinone pool and monitor pH changes in the lumen. Due to the spatial resolution of fluorescent microscopy, hundreds of liposomes can be measured simultaneously while the enzyme content can be scaled down to a single enzyme or transporter per liposome. The respective single enzyme analysis can reveal patterns in the enzyme functional dynamics that might be otherwise hidden by the behavior of the whole population. We include a description of a script for automated image analysis.

Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

Liposoma singole misurazioni per lo studio degli enzimi di membrana Proton-pompaggio usando microscopia fluorescente ed elettrochimica

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

Ricerca

Biochimica

In vitro Monitoring of Extracellular pH in Real-Time

Early accumulation of neutrophils (PMN) is a hallmark of acute intestinal inflammation. This acute inflammation is either resolved or progresses to chronic inflammation. Without efficient PMN clearance at sites of infiltration, PMN can accumulate and contribute to chronic inflammatory conditions, including the intestinal diseases ulcerative colitis (UC) and Crohn's Disease (CD). The pH in the distal colon in individuals with active UC can range between a pH of 5 and 6, whereas healthy individuals maintain colonic pH in the range of 6.8-7.4. Extracellular pH has been shown to influence both intestinal epithelial cells and the infiltrating immune cells. More specifically, extracellular acidosis significantly impacts PMN. At pH below 6.5, there are increases in the production of H2O2, inhibition of apoptosis, and increases in the functional lifespan of PMN. Given the significant presence of PMN and extracellular acidification at sites of inflammation, we developed a novel model that allows for the monitoring of extracellular pH during PMN transepithelial migration in real time. Here, we describe this model and how it can be utilized to measure both the apical and basal pH during PMN trafficking. This model can be utilized to monitor extracellular pH under a wide range of conditions; including, hypoxia, PMN transepithelial migration, and for extended periods of time.

In vitro Monitoring of Extracellular pH in Real-Time

This article represents a useful in vitro assay to measure changes in extracellular pH during neutrophil (PMN) transepithelial migration (TEM)

Early accumulation of neutrophils (PMN) is a hallmark of acute intestinal inflammation. This acute inflammation is either resolved or progresses to ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

Misurare phagosome pH Raziometrico microscopia a fluorescenza

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

Immunologia e infezioni

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement.

This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.

Imaging dei neutrofili phagosome e nel citoplasma utilizzo di un indicatore di pH Raziometrico

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular ...

Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, periodontitis, mucosal infections and, most notably, early childhood caries. In all of these conditions, the pH in the biofilm matrix impacts microbe-host interactions and thus the disease progression. The present protocol describes a confocal microscopy-based method to monitor pH dynamics inside cross-kingdom biofilms comprising Candida albicans and Streptococcus mutans. The pH-dependent dual-emission spectrum and the staining properties of the ratiometric probe C-SNARF-4 are exploited to determine drops in pH in extracellular areas of the biofilms. Use of pH ratiometry with the probe requires a meticulous choice of imaging parameters, a thorough calibration of the dye, and careful, threshold-based post-processing of the image data. When used correctly, the technique allows for the rapid assessment of extracellular pH in different areas of a biofilm and thus the monitoring of both horizontal and vertical pH gradients over time. While the use of confocal microscopy limits Z-profiling to thin biofilms of 75 &#181;m or less, the use of pH ratiometry is ideally suited for the noninvasive study of an important virulence factor in cross-kingdom biofilms.

The protocol describes the cultivation of cross-kingdom biofilms consisting of Candida albicans and Streptococcus mutans and presents a confocal microscopy-based method for the monitoring of extracellular pH inside these biofilms.

Monitoraggio del pH extracellulare nei biofilm tra regni incrociati utilizzando la microscopia Confocal

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, ...

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH are highly regulated in yeast cells and occupy a central role in overall pH homeostasis. We describe protocols for ratiometric measurement of pH in vivo using pH-sensitive fluorophores localized to the vacuole or cytosol. Vacuolar pH is measured using BCECF, which localizes to the vacuole in yeast when introduced into cells in its acetoxymethyl ester form. Cytosolic pH is measured with a pH-sensitive GFP expressed under control of a yeast promoter, yeast pHluorin. Methods for measurement of fluorescence ratios in yeast cell suspensions in a fluorimeter are described. Through these protocols, single time point measurements of pH under different conditions or in different yeast mutants have been compared and changes in pH over time have been monitored. These methods have also been adapted to a fluorescence plate reader format for high-throughput experiments. Advantages of ratiometric pH measurements over other approaches currently in use, potential experimental problems and solutions, and prospects for future use of these techniques are also described.

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).

Misurazione di pH vacuolare e di Cytosolic<em&gt; In Vivo</em&gt; In Lievito Cell Sospensioni

Vacuolar and  cytosolic pH are highly regulated in yeast cells and occupy a central role in  overall pH homeostasis. We describe  protocols for ...

Biologia

Measuring the pH, Redox Chemistries, and Degradative Capacity of Macropinosomes using Dual-Fluorophore Ratiometric Microscopy

In recent years, the field of macropinocytosis has grown rapidly. Macropinocytosis has emerged as a central mechanism by which innate immune cells maintain organismal homeostasis and immunity. Simultaneously, and in contrast to its homeostatic role, it can also drive various pathologies, including cancer and viral infections. Unlike other modes of endocytosis, the tools developed for studying the maturation of macropinosomes remain underdeveloped. Here the protocol describes newly developed tools for studying the redox environment within the lumen of early and maturing macropinosomes. Methodologies for using ratiometric fluorescence microscopy in assessing the pH, production of reactive oxygen species, and the degradative capacity within the lumen of individual macropinosomes in live cells are described. Single organelle measurements offer the advantage of revealing spatiotemporal heterogeneity, which is often lost with population-based approaches. Emphasis is placed on the basic principles of dual fluorophore ratiometric microscopy, including probe selection, instrumentation, calibration, and single-cell versus population-based methods.

We describe protocols for measuring pH, oxidative events, and protein digestion in individual macropinosomes in live cells. An emphasis is placed on dual-fluorophore ratiometric microscopy and the advantages it offers over population-based techniques.

Misurazione del pH, delle sostanze chimiche redox e della capacità degradativa dei macropinosomi utilizzando la microscopia a doppio fluoroforo

In recent years, the field of macropinocytosis has grown rapidly. Macropinocytosis has emerged as a central mechanism by which innate immune cells ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

Le domande di pHluorin per quantitativa, Cinetica e high-throughput analisi di endocitosi in germogliamento lievito

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

Misurazione simultanea di mitocondriale calcio e mitocondriale potenziale di membrana in cellule vive di Microscopia fluorescente

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

Utilizzando Recettori pHluorin-tag per il monitoraggio subcellulare localizzazione e il traffico

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

La misurazione simultanea di pH in endocitosi e citosoliche scomparti nelle cellule viventi usando Microscopia confocale

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In vitro Monitoring of Extracellular pH in Real-Time

Misurare phagosome pH Raziometrico microscopia a fluorescenza

Imaging dei neutrofili phagosome e nel citoplasma utilizzo di un indicatore di pH Raziometrico

Monitoraggio del pH extracellulare nei biofilm tra regni incrociati utilizzando la microscopia Confocal

Misurazione di pH vacuolare e di Cytosolic

Misurazione del pH, delle sostanze chimiche redox e della capacità degradativa dei macropinosomi utilizzando la microscopia a doppio fluoroforo

Le domande di pHluorin per quantitativa, Cinetica e high-throughput analisi di endocitosi in germogliamento lievito

Misurazione simultanea di mitocondriale calcio e mitocondriale potenziale di membrana in cellule vive di Microscopia fluorescente

Utilizzando Recettori pHluorin-tag per il monitoraggio subcellulare localizzazione e il traffico