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. Preparação das soluções de calibração para pH celular <ol><li> Adicionar os seguintes compostos de cinco tubos cónicos de 50 ml separados para preparar soluções a 5 para ser utilizados para a calibração: <ul><li> De NaCl (1 ml de 1 M de NaCl) (NaCl 1 M = 0,58 g em 10 ml de H 2 0) </li><li> KCl (6,75 ml de 1 M de KCl) (1 M KCl = 0,75 g em 10 ml de H 2 0) </li><li> Glucose (1 ml de 1 M de D-glicose) (1M de D-Glucose = 1,80 g em 10 ml de H 2 0) </li><li> MgS0 4 (0,05 ml de 1 M de MgSO4) (1 M MgSO4 = 1,20 g em 10 ml de H 2 0) </li><li> HEPES 20 mM (1 ml de HEPES 1M) (HEPES 1 M = 2,38 g em 10 ml de H 2 0) </li><li> CaCl2 (0,05 ml de 1M de CaCl2) (1 M CaCl2 = 1,47 g em 10 ml de H 2 0) Prepare todas as soluções em duas vezes destilada H 2 O (DDH 2 O). </li></ul></li><li> Complete cada solução com 35 ml de ddH2O e misturar com um agitador magnético até entãolução é alcançado. </li><li> Ajustar o pH de cada solução com 1 N de KOH ou HCl 1 N para se obter os valores de 5,5, 6,0, 6,5, 7,0 e 7,5 de pH. Em seguida, ajustar o volume com água desionizada até um volume final de 50 ml. </li><li> Medida 5 mg de nigericina e adicioná-lo ao 670 ul de EtOH absoluto para se preparar uma solução de 10 mM de nigericina estoque. Atenção: nigericina é altamente tóxico e deve ser manuseado com muito cuidado (luvas, óculos de segurança e máscara). Misture a solução por inversão até a nigericina esteja completamente dissolvido. </li><li> Adicionar 0,05 ml de nigericina 10 mM (concentração final 10 uM) a cada solução de calibração. Nigericina é um ionóforo que aumenta a permeabilidade da membrana da célula e causa de equilíbrio do pH intracelular e extracelular, na presença de uma concentração de despolarização de K + extracelular. </li><li> Soluções de calibração podem ser armazenados até 1 mês a 4 ° C </li></ol> 2. Preparação celular <ol><li> Sob um Safet biológicay capuz, as células de semente de 35 milímetros x 10 milímetros em pratos de cultura de 2 ml de meio de crescimento suplementado com 10% de soro fetal bovino e antibióticos. Utilizar a quantidade de células necessárias para assegurar cerca de 50% de confluência após 24 hr. NOTA: Em nossos experimentos, usamos a linha de células HT-1080 e placa 1 x 10 5 células por placa de cultura. </li><li> Determinar o número de pratos de cultura necessárias para a 5 pratos de cultura adicionais experimento e placa a ser usada para a calibração. </li><li> Pratos de cultura em lugar de um de 37 ° C / 5% de CO2 durante pelo menos 24 horas. </li></ol> 3. Preparação de Sondas de pH sensores <ol><li> Prepare uma solução estoque 1M da sonda fluorescente pH 8 hidroxipireno-1, 3,6 trissulfónico, sal trissódico (HPTS / piranina) em DDH 2 a 0. Esta solução deve ser armazenada à temperatura ambiente e protegida da luz. </li><li> Prepara-se uma solução de reserva de 1 mM de 5 - (e-6)-carboxil seminaphthorhodafluor acetoximetil éster de etilo (C-SNARF-01:00; chamado Thereaepois SNARF-1) através da dissolução de 1 mg de SNARF-1 em 1,76 ml de DMSO. Misture bem por pipetagem repetida. SNARF-1 solução deve ser armazenada a -20 ° C e protegida da luz. </li></ol> 4. Labeling celular <ol><li> Dezasseis horas antes de imagem ao vivo de células, adicionar 2 ml de solução de estoque 1M HPTS (concentração final 1 mM) para cada placa de cultura contendo 2 ml de meio completo. </li><li> Retorne os pratos de cultura para a incubadora. A sonda HPTS é célula-impermeant, portanto, a etiqueta organelas a sonda deve ser tomada por pinocitose. Esta condição implica que as incubações das células com HPTS são realizadas em meio de cultura suplementado com soro. </li><li> Após a incubação com HPTS, lavar as células duas vezes com PBS estéril e adicionar 2 ml de meio isento de soro, durante pelo menos 2 horas. A incubação com SNARF-1 tem de ser realizada em meio isento de soro, porque o soro pode impedir a absorção da sonda. </li><li> Adicionam-se 10 ul de uma SNARF-1 de solução stock a 1 mM para se obter afinal concentração de 5 uM e permitir que as células a incubar durante 20 minutos a 37 ° C, sob luz fraca. C-1-SNARF concentração e tempo de incubação pode variar de acordo com o tipo de célula. </li><li> No final da incubação, lavar as células duas vezes em PBS estéril e adicionar 2 ml de meio isento de soro. </li></ol> 5. Adição de pH moduladores <ol><li> Para avaliar se o método pode detectar alterações de pH nas células, diferentes reagentes que modulam o pH intracelular pode ser usado: <ul><li> Bafilomicina A1, um inibidor específico da V-ATPase 5 </li><li> Isoforma permutador Na + / H + 1 (NHE-1) 6 </li><li> NH4Cl, uma base fraca que aumenta o pH intracelular 7 </li></ul><ol><li> Preparar soluções estoque de reagentes mencionados acima: <ul><li> Bafilomicina A1: acrescentar 10 ug de bafilomicina A1 a 160 ul de DMSO para se obter uma solução stock de 100 uM </li><li> EIPA: adicionar 25 mg de EIPA para 1,67 ml de metanol a obtina concentração final de 50 mM </li><li> NH4Cl: dissolver 0,53 g de cloreto de amónio em 10 ml de ddH2O para obter uma concentração final de 1 mM. </li></ul> Nota: Todas as soluções de reserva deve ser mantida a 4 ° C, excepto para bafilomicina A1, que deve ser armazenada a -20 ° C e protegida da luz. </li><li> Em pratos de cultura separados, adicionar um dos reagentes, tal como descrito abaixo: <ul><li> 2 ul de 100 uM bafilomicina A1 (concentração final 100 nM) </li><li> 1 ul de EIPA 50 mM (concentração final 25 uM) </li><li> 40 ul de 1M de NH 4 Cl (concentração final 20 mM) </li></ul></li><li> Incubar as células durante 30 min a 37 ° C. </li></ol></li><li> Vá para a aquisição de imagem. </li></ol> 6. Especificações do Microscópio de imagem de células vivas e configurações de aquisição <ol><li> Realize imagens ao vivo de células usando uma objetiva de 40X montado em um invertido digitalização microscopia confocal espectralpe equipado com um estágio XY motorizada, uma câmara de vidro que acomoda placas de cultura de fundo em um ambiente controlado (temperatura, umidade, CO 2) fase incubadora. </li><li> O instrumento confocal (Olympus FV1000) usada aqui está equipado com: <ul><li> Um laser de diodo 405 nm </li><li> Um laser de argônio de 40 mW (458 nm, 488 nm, 515 nm) </li><li> Um laser neon hélio (543 nm) </li><li> Objetivo de fluorita de 10x, NA0.4 </li><li> Um objectivo LUCPLFLN 40X, NA0.60 </li><li> Um objectivo óleo PLAPONSC PLANO APO 60X, NA1.4 </li><li> Um espelho dicróico 405/458/488/543 DM </li><li> A SDM 560 e um SDM 640 filtros dicróicas </li><li> Um filtro de barreira BA655-755 </li><li> A BS20/80 divisor de feixe </li><li> O software de análise de dados para a aquisição e análise de imagem, </li></ul></li><li> Uma vez que a incubadora estágio tem aquecido até 37 ° C e o microscópio está configurado, coloque um prato de cultura previamente incubadas com HPTS e SNARF-1 para a câmara de estágio imagem confocal. </li><li> Aepois de localizar uma área de interesse, ajuste as configurações de aquisição de software Figura 1. <ul><li> Configure dois conjuntos de canais virtuais. </li><li> Iniciar a primeira fase que corresponde ao HPTS excitação a 405 nm e coleta de emissão em 505-525 nm e SNARF-1 de excitação em 543 nm e coleta de emissão em 570-600 nm. </li><li> Inicie a segunda fase que corresponde ao HPTS excitação em 458 nm e coleta de emissão em 505-525 nm e SNARF-1 de excitação em 543 nm e coleta de emissão em 640 - 650 nm. </li><li> A abertura confocal está definido para 175 um por padrão e abertura ideal precisa ser configurado manualmente em 300 mm. </li><li> Tome Imagem por empilhamento de série secções ópticas horizontais (800 x 800 pixels) de 0,4 m de espessura. </li></ul></li></ol> NOTA: uma objetiva de 40X é útil para observar várias células simultaneamente e funciona bem para a visão organela. No entanto, a resolução espacial e intensidade do sinal são better conseguido usando uma objetiva de óleo 60X. Além disso, não há excitação ou de emissão de conversa cruzada entre os dois corantes. 7. Calibração de Sondas de pH de sensoriamento <ol><li> Antes da calibração, remover o meio da placa de cultura previamente incubadas com HPTS e SNARF-1, lavar duas vezes com PBS estéril e, em seguida, adicionar 2 ml de uma das cinco soluções de calibração. </li><li> Coloque o prato de cultura na câmara de estágio confocal. </li><li> Vá para a aquisição de imagens de lapso de tempo de acordo com as seguintes definições: <ol><li> Defina o número de imagens e tempo de duração do intervalo para o experimento. NOTA: Normalmente usamos um intervalo de 1 min entre as imagens por até 30 min. </li><li> Programe o obturador para fechar entre a aquisição de cada imagem. </li></ol></li><li> Gravar a intensidade de fluorescência por minuto em um software de folha de cálculo e de determinar o momento em que a intensidade de fluorescência é constante. PH intracelular é geralmente estável depois de 15 - 20 min. </li><li> REPEAT os passos 7.1 a 7.4 usando um novo prato de cultura para cada solução de calibração restante. </li></ol> 8. Aquisição de Dados <ol><li> Uma vez que a calibração foi feito, adquirir dados de amostra 4-5 campos escolhidos aleatoriamente contendo 15-20 células por campo. Duas imagens são obtidas para cada campo: um para rotulagem HPTS (vesículas) e o segundo para SNARF-1 rotulagem (citosol). </li><li> A fluorescência de fundo (zona extracelular) é subtraído digitalmente imagens fluorescentes de ambas as sondas. Vesículas rotulados e citosol de suas respectivas imagens armazenadas são selecionados usando uma máscara binária. Para cada sonda de pH-sensing, intensidades de fluorescência são medidos em uma escala numérica (0-4,095 escala de nível de cinza). A intensidade média de todos os pixels na organelo ou citoplasma é calculada e os valores são utilizados para calcular os rácios de fluorescência. </li></ol> 9. Data Analysis <ol><li> Vários pacotes de software estão comercialmente disponíveis para a análise de dados. IstoSoftware FluoView. </li><li> Em uma planilha, valores de registro e calcular índices de fluorescência da seguinte forma: No caso de HPTS, R = (F 458 nm / F 405 nm) e, no caso de SNARF-1, R = (F 644 nm / F 584 nm ). </li><li> Plotar os dados usando o software de gráficos científica como GraphPad Prism. A trama deve representar proporções de fluorescência (eixo Y) como uma função do pH (eixo X). Ajuste de curva é obtida por meio de regressão não-linear. </li><li> Recolhe intensidades de fluorescência das amostras, conforme descrito no passo 8.2 e calcular rácios de fluorescência. Use as curvas de calibração para transformar proporções em 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.

Medição de pH quantitativa em imagens ao vivo de células de diferentes compartimentos celulares é necessário para medir com precisão as variações de pH em respostas das células aos desafios externos. No entanto, um dos principais obstáculos que resta é fácil e visam especificamente compartimentos celulares. A este respeito, alguns estudos puseram em evidência o uso de HPTS como um indicador de pH intracelular introduzido nas células por electroporação ou microinjecção 8-10. Estas técnicas s...

<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.

Para a medição simultânea de pH em ambos os compartimentos da célula intracelular e endossomal / lisossomal, foram usadas as sondas de pH de detecção fluorescente raciométrica Snarf-1 e HPTS. SNARF-1 é restrita ao compartimento citosólico enquanto HPTS permite a medição de o compartimento endossomal / lisossomal raciométrica pH. Carregamento celular com HPTS para 16 horas permite a sua localização seletiva em endossomas / 4 lisossomos. Um exemplo típico de fluorescência registada a partir de c...

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.

Medição simultânea em pH endocítica e citossólicos Compartimentos em células vivas usando microscopia 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

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JoVE Journal

Biology

14.9K visualizações.  Université de Sherbrooke. Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells. 

Vídeo: 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.

Medições de lipossoma único para o estudo das enzimas de membrana de bombeamento de prótons usando microscopia fluorescente e eletroquímica

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

Bioquímica

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.

Medindo phagosome pH Raciometrico por microscopia de fluorescência

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

Imunologia e Infecção

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 o neutrófilos fagossoma e citoplasma utilizando um indicador de pH Ratiometric

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.

Monitoramento de pH extracelular em biofilmes transversais usando 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).

Medição do valor pH vacuolar e Cytosolic<em&gt; In Vivo</em&gt; Em Suspensions célula de levedura

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.

Medindo o pH, As Químicas Redox e a Capacidade Degradativa dos Macropinossos usando microscopia multiscopia memétrica de proporção de fluorofora dupla

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.

Aplicações de pHluorin para Quantitative, Kinetic e de alta capacidade: Análise de endocitose em brotamento Yeast

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.

Medição simultânea de mitocondrial de cálcio e mitocondrial potencial de membrana em células vivas por 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.

Utilizando Receptores pHluorin-tag para monitorar subcelular Localização e Tráfico

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

Medição simultânea em pH endocítica e citossólicos Compartimentos em células vivas usando microscopia confocal

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In vitro Monitoring of Extracellular pH in Real-Time

Medindo phagosome pH Raciometrico por microscopia de fluorescência

Imaging o neutrófilos fagossoma e citoplasma utilizando um indicador de pH Ratiometric

Monitoramento de pH extracelular em biofilmes transversais usando microscopia confocal

Medição do valor pH vacuolar e Cytosolic

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Aplicações de pHluorin para Quantitative, Kinetic e de alta capacidade: Análise de endocitose em brotamento Yeast

Medição simultânea de mitocondrial de cálcio e mitocondrial potencial de membrana em células vivas por Microscopia fluorescente

Utilizando Receptores pHluorin-tag para monitorar subcelular Localização e Tráfico