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. Preparación de las soluciones de calibración pH celular <ol><li> Agregue los siguientes compuestos de cinco tubos de 50 ml cónicos separados para preparar 5 soluciones que se utilizarán para la calibración: <ul><li> De NaCl (1 ml de 1 M de NaCl) (1 M de NaCl = 0,58 g en 10 ml de H 2 0) </li><li> KCl (6,75 ml de KCl 1 M) (1 M KCl = 0,75 g en 10 ml de H 2 0) </li><li> La glucosa (1 ml de 1 M de D-glucosa) (1M D-glucosa = 1,80 g en 10 ml de H 2 0) </li><li> MgS0 4 (0,05 ml de 1 M MgSO4) (1 M MgSO4 = 1,20 g en 10 ml de H 2 0) </li><li> HEPES 20 mM (1 ml de HEPES 1 M) (1 M HEPES = 2,38 g en 10 ml de H 2 0) </li><li> CaCl2 (0,05 ml de CaCl2 1 M) (1 M de CaCl 2 = 1,47 g en 10 ml de H 2 0) Preparar todas las soluciones en doble destilada H 2 O (ddH2O). </li></ul></li><li> Complete cada solución con 35 ml de ddH2O y mezclar con un agitador magnético hasta que por lolución se logra. </li><li> Ajuste el pH de cada solución con KOH 1 N o HCl 1 N para obtener valores de pH de 5,5, 6,0, 6,5, 7,0 y 7,5. A continuación, ajuste el volumen con agua desionizada hasta un volumen final de 50 ml. </li><li> Medida 5 mg de nigericina y agregarlo a 670 l de EtOH absoluto para preparar a 10 mM nigericina solución madre. Precaución: nigericina es altamente tóxica y debe manejarse con mucho cuidado (guantes, gafas de seguridad y mascarilla). Mezclar la solución por inversión hasta que la nigericina se haya disuelto completamente. </li><li> Añadir 0,05 ml de 10 mM de nigericina (concentración final 10 mM) a cada solución de calibración. Nigericina es un ionóforo que aumenta la permeabilidad de la membrana celular y causa de equilibrado del pH intracelular y extracelular en presencia de una concentración de despolarización de K + extracelular. </li><li> Soluciones de calibración se pueden almacenar hasta 1 mes a 4 ° C </li></ol> 2. Preparación de la célula <ol><li> Bajo un safet biológicaY capó, en células de semillas 35 mm de placas de cultivo de 10 mm x en 2 ml de medio de crecimiento suplementado con suero bovino fetal al 10% y antibióticos. Utilice la cantidad de células necesaria para asegurar aproximadamente el 50% de confluencia después de 24 horas. NOTA: En nuestros experimentos, utilizamos la línea celular HT-1080 y 1 x 10 5 células por placa de cultivo en placa. </li><li> Determinar el número de placas de cultivo necesarios para el experimento y la placa 5 placas de cultivo adicionales que se utilizarán para la calibración. </li><li> Platos sitúan la cultura en el C / 5% de CO 2 incubadora a 37 º durante al menos 24 horas. </li></ol> 3. Preparación de sondas de sensor de pH <ol><li> Preparar una solución madre 1 M de la sonda de pH fluorescente 8-hidroxipireno-1, 3,6 trisulfónico, sal trisódica (HPTS / piranina) en ddH 2 0. Esta solución debe ser almacenada a temperatura ambiente y protegido de la luz. </li><li> Preparar una solución madre de 1 mM 5 - (y-6)-carboxilo seminaphthorhodafluor acetoximetil éster de acetato (C-SNARF-1 am; llamado thereâespués de SNARF-1) por disolución de 1 mg de SNARF-1 en 1,76 ml de DMSO. Mezcle bien con la pipeta repetido. SNARF-1 solución deberá conservarse a -20 ° C y protegido de la luz. </li></ol> 4. Etiquetado Celular <ol><li> Dieciséis horas antes de imágenes de células vivas, añadir 2 l de solución madre 1 M de HPTS (concentración final 1 mM) a cada placa de cultivo que contiene 2 ml de medio completo. </li><li> Vuelva a poner las placas de cultivo a la incubadora. La sonda HPTS es celular-impermeant, por lo tanto, a la etiqueta organelos la sonda debe ser tomado por pinocitosis. Esta condición requiere que las incubaciones de células con HPTS se llevan a cabo en medio de cultivo suplementado con suero. </li><li> Tras la incubación con HPTS, se lavan las células dos veces con PBS estéril y añadir 2 ml de medio libre de suero durante al menos 2 horas. La incubación con SNARF-1 necesita ser realizada en medio libre de suero, porque el suero puede prevenir la absorción de la sonda. </li><li> Añadir 10 l de una SNARF-1 de solución madre 1 mM para obtener afconcentración inal de 5 M y permiten a las células se incuban durante 20 minutos a 37 ° C, bajo luz tenue. C-SNARF-1 concentración y tiempo de incubación pueden variar según el tipo de célula. </li><li> Al final de la incubación, lavar las células dos veces en PBS estéril y añadir 2 ml de medio libre de suero. </li></ol> 5. La adición de pH Moduladores <ol><li> Para evaluar si el método puede detectar cambios de pH en las células, diferentes reactivos que modulan el pH intracelular se pueden utilizar: <ul><li> Bafilomicina A1, un inhibidor específico de la V-ATPasa 5 </li><li> De Na + / H + intercambiador de isoforma 1 (NHE-1) 6 </li><li> NH 4 Cl, una base débil que aumenta intracelular pH 7 </li></ul><ol><li> Preparar las soluciones madre de los reactivos mencionados anteriormente: <ul><li> Bafilomicina A1: añadir 10 g de bafilomicina A1 a 160 l de DMSO para obtener una solución madre de 100 mM </li><li> EIPA: añadir 25 mg de EIPA a 1,67 ml de metanol para obtena concentración final de 50 mM </li><li> NH4Cl: disolver 0,53 g de cloruro de amonio en 10 ml de ddH2O para obtener una concentración final de 1 mM. </li></ul> Nota: Todas las soluciones madre deben mantenerse a 4 ° C con excepción de bafilomicina A1, que deben ser almacenados a -20 ° C y protegido de la luz. </li><li> En placas de cultivo separadas, agrega uno de los reactivos como se indica a continuación: <ul><li> 2 l de 100 mM bafilomicina A1 (concentración final 100 nM) </li><li> 1 l de 50 mM EIPA (concentración final 25 mM) </li><li> 40 l de 1 M NH4Cl (concentración final 20 mM) </li></ul></li><li> Se incuban las células durante 30 min a 37 ° C. </li></ol></li><li> Proceder a la adquisición de imágenes. </li></ol> 6. Especificaciones del microscopio Células vivas imágenes y ajustes de adquisición <ol><li> Realizar imágenes de células vivas utilizando un objetivo de 40x montada en un escaneo confocal espectral invertida microscópicape equipado con una platina motorizada XY, una cámara que acomoda vidrio placas de cultivo de fondo en un ambiente controlado (temperatura, humedad, CO 2) etapa incubadora. </li><li> El instrumento confocal (Olympus FV1000) usado aquí está equipado con: <ul><li> Un láser de diodo de 405 nm </li><li> Un láser de argón de 40 mW (458 nm, 488 nm, 515 nm) </li><li> Un láser de helio-neón (543 nm) </li><li> Fluorita objetivo 10X, NA0.4 </li><li> Un objetivo LUCPLFLN 40X, NA0.60 </li><li> Un objetivo aceite PLAPONSC PLAN APO 60X, NA1.4 </li><li> Un espejo dicroico 405/458/488/543 DM </li><li> Un SDM 560 y un SDM 640 filtros dicroicos </li><li> Un filtro de barrera BA655-755 </li><li> A BS20/80 divisor de haz </li><li> Software de análisis de datos para la adquisición y análisis de imágenes, </li></ul></li><li> Una vez que la etapa incubadora se ha calentado a 37 ° C y el microscopio está establecido, coloque una placa de cultivo previamente incubados con HPTS y SNARF-1 en la cámara de imagen confocal etapa. </li><li> Laespués de la localización de un área de interés, ajustar la configuración de adquisición de software Figura 1. <ul><li> La creación de dos conjuntos de canales virtuales. </li><li> Iniciar la primera fase que corresponde a HPTS excitación a 405 nm y recogida de emisión a 505-525 nm y SNARF-1 excitación a 543 nm y recogida de emisión a 570-600 nm. </li><li> Iniciar la segunda fase que corresponde a HPTS excitación a 458 nm y recogida de emisión a 505-525 nm y SNARF-1 excitación a 543 nm y recogida de emisión a 640-650 nm. </li><li> La apertura confocal se establece en 175 micras por defecto y apertura óptimos debe configurarse manualmente a 300 micras. </li><li> Tome imagen apilando secciones ópticas horizontales en serie (800 x 800 píxeles) de 0,4 m de espesor. </li></ul></li></ol> NOTA: Un objetivo de 40X es útil observar varias celdas a la vez y funciona bien para la visualización orgánulo. Sin embargo, la resolución espacial y la intensidad de la señal son better logra utilizando un objetivo de aceite de 60X. Además, no hay ninguna excitación o de emisión de la diafonía entre los dos colorantes. 7. Calibración de sondas de pH con sensor <ol><li> Antes de la calibración, retire medio de la placa de cultivo previamente incubadas con HPTS y SNARF-1, lavar dos veces con PBS estéril y luego añadir 2 ml de una de las cinco soluciones de calibración. </li><li> Coloque la placa de cultivo en la cámara etapa confocal. </li><li> Proceda a la adquisición de la imagen de lapso de tiempo de acuerdo a los siguientes parámetros: <ol><li> Configure el número de imágenes y de los tiempos de duración de intervalo para el experimento. NOTA: Por lo general utilizan un intervalo de 1 minuto entre las imágenes para un máximo de 30 min. </li><li> Programar el obturador para cerrar entre la adquisición de cada imagen. </li></ol></li><li> Anote la intensidad de fluorescencia por minuto en un software de hoja de cálculo y determinar el momento en el que la intensidad de fluorescencia es constante. PH intracelular es generalmente estable después del 15 - 20 min. </li><li> REPEAT pasos 7.1 a 7.4 usando una nueva placa de cultivo para cada solución de calibración restante. </li></ol> 8. Adquisición de Datos <ol><li> Una vez que la calibración se ha hecho, la adquisición de datos de la muestra 4-5 campos elegidos al azar que contienen 15 a 20 células por campo. Dos imágenes se obtienen para cada campo: uno para el etiquetado de HPTS (vesículas) y la segunda para SNARF-1 etiquetado (citosol). </li><li> La fluorescencia de fondo (zona extracelular) se resta digitalmente de imágenes fluorescentes de ambas sondas. Vesículas etiquetadas y citosol de sus respectivas imágenes almacenadas se seleccionan usando una máscara binaria. Para cada sonda de pH con sensor de intensidad de fluorescencia se miden en una escala numérica (0-4,095 escala de nivel de gris). Una intensidad media para todos los píxeles en el orgánulo o citoplasma se calcula y valores se utilizan para calcular los coeficientes de fluorescencia. </li></ol> 9. Análisis de Datos <ol><li> Distintos paquetes de software disponibles en el mercado para el análisis de datos. IeSoftware Fluoview. </li><li> En una hoja de cálculo, valores de registro y cálculo de coeficientes de fluorescencia de la siguiente manera: En el caso de HPTS, R = (F 458 nm / F 405 nm) y, en el caso de SNARF-1, R = (F 644 nm / F 584 nm ). </li><li> Grafica los datos utilizando software de gráficos científico como GraphPad Prism. La trama debe representar coeficientes de fluorescencia (eje Y) como una función del pH (eje X). El ajuste de curvas se consigue mediante regresión no lineal. </li><li> Recoger la intensidad de fluorescencia de las muestras como se describe en el paso 8.2 y calcular los coeficientes de fluorescencia. Utilice las curvas de calibración para transformar relaciones en valores de 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.

Es necesaria la medición cuantitativa de pH en imágenes de células vivas de diferentes compartimentos celulares para medir con precisión las variaciones de pH en las respuestas celulares a los desafíos externos. Sin embargo, uno de los principales obstáculos que queda es para apuntar fácilmente y, específicamente, los compartimentos celulares. A este respecto, algunos estudios han puesto de relieve el uso de HPTS como un indicador de pH intracelular introducido en las células mediante electroporación o microin...

<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.

Para la medición simultánea de pH en tanto intracelular y compartimentos celulares endosomal / lisosomales, se utilizaron las sondas fluorescentes de pH de detección ratiometric SNARF-1 y HPTS. SNARF-1 está limitada al compartimiento citosólico de HPTS mientras que permite la medición del pH radiométrica del compartimiento endosomal / lisosomal. Carga celular con HPTS durante 16 horas permite su localización selectiva en endosomas / lisosomas 4. Un ejemplo típico de fluorescencia registrada a partir ...

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.

Medición pH simultánea en Endocítica y citosólicas compartimentos en las células vivas usando microscopía confocal

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 vistas.  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

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Basado en la fluorescencia de medición de la entrada de calcio de la tienda que funciona en las células vivas: a partir de células cancerosas cultivadas de fibra muscular esquelética

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Investigación

Biología

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).

La medición de pH vacuolar y citosólica<em&gt; En Vivo</em&gt; En suspensiones de células de levadura

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

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

Analizando Craneofacial en la morfogénesis de pez cebra Utilizando 4D Microscopía Confocal

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

En Tracking Clonal vivo de madre hematopoyéticas y células progenitoras Marcado por cinco proteínas fluorescentes utilizando confocal y Microscopía Multifotónica

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Las mediciones del calcio citosólico en las células epiteliales renales mediante citometría de flujo

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

-Alta resolución de imagen de lapso de tiempo y análisis automatizado de los microtúbulos en la dinámica de estar umbilical humana Las células endoteliales de vena

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

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.

Medición simultánea de calcio mitocondrial y mitocondrial potencial de membrana en células vivas por microscopía fluorescente

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

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Una interfaz gráfica de usuario para el seguimiento asistido por software de la concentración de proteínas en protones dinámicos celulares

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

Sondeo de mRNA cinética en el espacio y el tiempo en Escherichia coli usando hibridación de fluorescencia de una sola molécula de dos colores

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...

Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional (x, y, z, and &lambda;) Hyperspectral FRET Imaging and Analysis

Cyclic AMP is a second messenger that is involved in a wide range of cellular and physiological activities. Several studies suggest that cAMP signals are compartmentalized, and that compartmentalization contributes to signaling specificity within the cAMP signaling pathway. The development of F&#1255;rster resonance energy transfer (FRET) based biosensors has furthered the ability to measure and visualize cAMP signals in cells. However, these measurements are often confined to two spatial dimensions, which may result in misinterpretation of data. To date, there have been only very limited measurements of cAMP signals in three spatial dimensions (x, y, and z), due to the technical limitations in using FRET sensors that inherently exhibit low signal to noise ratio (SNR). In addition, traditional filter-based imaging approaches are often ineffective for accurate measurement of cAMP signals in localized subcellular regions due to a range of factors, including spectral crosstalk, limited signal strength, and autofluorescence. To overcome these limitations and allow FRET-based biosensors to be used with multiple fluorophores, we have developed hyperspectral FRET imaging and analysis approaches that provide spectral specificity for calculating FRET efficiencies and the ability to spectrally separate FRET signals from confounding autofluorescence and/or signals from additional fluorescent labels. Here, we present the methodology for implementing hyperspectral FRET imaging as well as the need to construct an appropriate spectral library that is neither undersampled nor oversampled to perform spectral unmixing. While we present this methodology for measurement of three-dimensional cAMP distributions in pulmonary microvascular endothelial cells (PMVECs), this methodology could be used to study spatial distributions of cAMP in a range of cell types.

Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional x, y, z, and &amp;lambda; Hyperspectral FRET Imaging and Analysis

Due to inherent low signal-to-noise ratio (SNR) of F&#1255;rster resonance energy transfer (FRET) based sensors, measurement of cAMP signals has been challenging, especially in three spatial dimensions. Here, we describe a hyperspectral FRET imaging and analysis methodology that allows measurement of cAMP distribution in three spatial dimensions.

Medición de distribuciones de cAMP tridimensionales en células vivas utilizando imágenes y análisis FRET hiperespectrales de 4 dimensiones (x, y, z y λ)

Cyclic AMP is a second messenger that is involved in a wide range of cellular and physiological activities. Several studies suggest that cAMP signals are ...