The authors are financially supported by the Swiss National Science Foundation through an operating grant N&#176; 31003A-149566 (to N.D.), and The Sir Jules Thorn Charitable Overseas Trust through a Young Investigator Subsidy (to P.N.).

Declaración de Ética: Todas las manipulaciones animales se realizaron en estricta conformidad con las directrices del Comité de Investigación Animal de la Universidad de Ginebra. 1. Preparación de Metas fagocíticas <ol><li> Añadir 20 mg de zymosan se secó a 10 ml de solución salina tamponada con fosfato estéril (PBS). Vortex y el calor en un baño de agua hirviendo durante 10 minutos. Enfriar y centrifugar a 2000 xg durante 5 min. </li><li> Eliminar el sobrenadante, resuspender en 1 ml de PBS y se somete a ultrasonidos durante 10 minutos en un sonicador de baño de agua. Transferir 500 l a dos tubos de 1,5 ml. Centrifugar a 11.000 xg durante 5 min. </li><li> Repita el lavado con 500 l de PBS. El zymosan hervida puede ser en alícuotas, congelados y almacenados a -20 ° C. </li><li> Lave zimosán dos veces en 500 l recién preparados 0,1 M Na 2 CO 3, pH 9,3 que el anterior (paso 1.2). </li><li> Resuspender en 490 l después del lavado final. Añadir 10 l de 10 mg / ml de FITC disuelto en dimetil sulfoxide (DMSO), vórtice, cubrir con papel de aluminio, y se incuba con agitación durante 4 horas a temperatura ambiente. </li><li> Para quitar colorante no fijado, lávese que el anterior (paso 1.2), primero con 0,5 ml de DMSO + 150 l de PBS, y luego 0,5 ml de DMSO + 300 l de PBS, y luego 0,5 ml de DMSO + 450 l de PBS, a continuación PBS solo. </li><li> Cuente zimosán utilizando un hemocitómetro. Añadir 1 l de zymosan a 500 l de PBS, vórtice, a continuación, añadir 10 l hasta el borde de la cámara de hemocitómetro, donde se encuentra con el cubreobjetos se coloca sobre la rejilla central. Montar el hemocitómetro en un microscopio de campo brillante equipado con un objetivo 20X. </li><li> Centrarse en la esquina superior izquierda contiene 16 cuadrados y contar todas las partículas zymosan en esta área utilizando un contador de recuento mano. Repita con la esquina inferior derecha. Calcular la concentración zymosan usando la siguiente ecuación: concentración Zymosan = Cantidad / 2 x 500 x 10 4 zymosan / ml. Zimosán marcado puede ser en alícuotas, congelados y almacenados a -20 ° C. </li><li> Opsonizar el wi zimosán etiquetadoth un conejo anticuerpo anti-zymosan en 1: 100 dilución. Vortex e incubar durante 1 hora en un 37 ° C baño de agua. Lavar con 500 l de PBS que el anterior (paso 1.2). Nota: La sonicación es muy recomendable, sobre todo después de la opsonización, como zimosán tiende a formar montones que pueden hacer análisis más difíciles más adelante. Opsonización con 65% v / v de rata o de suero humano se puede utilizar como una alternativa. </li><li> Contar zymosan usando un hemocitómetro como se describe en los pasos 1,7-1,8, y se diluye hasta 100 x 10 6 partículas / ml. Opsonización con otros estimuladores fagocíticas, como complemento o no la opsonización, se puede realizar alternativamente. </li></ol> 2. Live Video Microscopía <ol><li> Aislar neutrófilos primarios de ratón como se describe en detalle en otra parte 38. <ol><li> También puede utilizar cualquier tipo de célula fagocítica. Mantener los neutrófilos en hielo en Roswell Park Memorial Institute (RPMI) 1640 suplementado con 5% de suero bovino fetal (v / v), 200 U de penicilina / streptomycin, a una concentración de 10 x 10 6 células / ml hasta que esté listo para su uso. Otros tipos de células se pueden sembrar 1-4 días antes. </li></ol></li><li> Añadir 2 x 10 5 (20 l) de los neutrófilos hacia el centro de un plato de fondo de vidrio 35 mm, se extendió la caída mediante la adición de 50 l de medio y se incuba a 37 ° C durante 5 min para permitir que las células se adhieran. </li><li> Añadir 1 ml de solución salina equilibrada de Hank (HBSS) se calentaron a 37 ° C que contiene la específica MPO hidrazida inhibidor 4-aminobenzoico (4-ABH, 10 M). Los inhibidores no específicos de la MPO, como azida de sodio (5 mM), también se pueden utilizar, pero se han sugerido recientemente para ejercer efectos no específicos adicionales en pH 39. </li><li> Montar el plato en un microscopio de fluorescencia de campo amplio equipado con un sistema de calefacción C 37 ° y un objetivo de aceite de 40X. Se incuban las células sin formación de imágenes durante 10 min para permitir que las células y los equipos se equilibren. </li><li> Ajuste la configuración de microscopio para adquirir una imagen de transmisión de campo brillante [phase o contraste de interferencia diferencial (DIC), si está disponible] con 440 nm o 490 nm de excitación y 535 nm de emisión cada 30 segundos. </li><li> Ajuste el tiempo de exposición y la frecuencia de adquisición de acuerdo con el sistema de microscopio para evitar el exceso de fotoblanqueo y fototoxicidad y en función de las preguntas experimentales se preguntó. Comience la adquisición de imágenes. </li><li> Después de 2 min, añadir 10 x 10 6 (50 l) opsonizado-FITC zymosan al centro del plato. Ajuste de destino: proporciones de células dependiendo del tipo de fagocito y de destino utilizado. </li><li> Continuar de imágenes para 30 min. Después de 30 minutos detener la adquisición de lapso de tiempo y comenzar un temporizador. Mueva el escenario y capturar 10 o más instantáneas en diferentes campos de visión de imagen otros fagosomas, todo dentro de 5 min. </li></ol> 3. Los experimentos de calibración y control <ol><li> Preparar las soluciones de calibración pH (recetas se describen en la Tabla 1) antes del día del experimento y congelar en alícuotas de 10 ml.Descongele las soluciones de calibración y caliente a 37 ° C en un baño de agua. Comprobar el pH con un medidor de pH y registrar el pH real de las soluciones. </li><li> Montar una bomba peristáltica en la parte inferior plato de cristal antes de la adquisición de la imagen y realizar microscopía de vídeo en directo como se describe anteriormente (sección 2). </li><li> Al final del período de adquisición de 30 min encienda la bomba para eliminar la solución dentro del plato, añadir 1 ml de la primera solución de calibración y detener la bomba. </li><li> Espere hasta que la señal se estabiliza, por lo general 5 min. Repita el procedimiento con cada solución de calibración. <ol><li> Realizar al menos una calibración por día experimental, y calibrar comenzando con el más alto pH y secuencial de trabajo a la más baja, así como a partir de la pH más bajo y trabajando de forma secuencial hacia el más alto. </li></ol></li><li> Como control, añadir 100 l de 100 mM de yoduro de propionium difenil inhibidor de la NADPH oxidasa (DPI) diluido en HBSS a las células en 900 l de HBSS en el plato de fondo de vidrio, yincubar durante 10 min. </li><li> Comience adquisición y añadir zimosán que el anterior (paso 2.7). Después de 15 min de la fagocitosis, añadir 10 l de 10 mM inhibidor de la V-ATPasa concanamycin A (ConcA) diluido en HBSS y continuar de imágenes para un adicional de 15 min. </li></ol> 4. Análisis <ol><li> Análisis de películas a intervalos y las instantáneas fagosoma. Nota: Las siguientes instrucciones son específicas para ImageJ. Paso a paso las instrucciones pueden variar ampliamente dependiendo del software utilizado, pero las funciones descritas en esta sección están disponibles en la mayoría de software de análisis de imagen profesional. <ol><li> Abra las imágenes con software de análisis de imagen profesional. Reste el fondo en el canal 490 dibujando una pequeña ROI cuadrado con la herramienta de selección poligonal cuadrados en una zona donde no hay células, y haciendo clic en Plugins&gt; BG Sustracción de retorno de la inversión. Luego cargue el mismo ROI en el canal 440 haciendo clic en Editar&gt; Cambios&gt; Restaurar selección y repetir. </li><li>Hacer una imagen de relación de 32 bits del canal de 490 dividido por el canal 440 haciendo clic en Proceso&gt; Calculadora Imagen ..., la selección de las imágenes apropiadas en los menús desplegables Image1 y Image2, luego seleccionando &quot;brecha&quot; en el menú desplegable de la Operación y comprobar el resultado casilla 32-bit (float). </li><li> Cambie la tabla de consulta de códigos de colores para un código de colores compatibles con relación haciendo clic en Imagen&gt; tablas de búsqueda&gt; Rainbow RGB. Umbral de la imagen relación de eliminar 0 e infinito píxeles haciendo clic en Imagen&gt; Ajustar&gt; Umbral .... Ajuste las barras de desplazamiento para que zimosán aparece en rojo y haga clic en Aplicar, asegurándose de que los valores de ajuste píxeles de fondo casilla NaN a se selecciona. </li><li> Combine esta relación pila con el canal de campo brillante en un compuesto de varios canales haciendo clic en Imagen&gt; Color&gt; Combinar Canales, y la selección de la imagen de campo brillante en el menú desplegable canal gris y la imagen de relación en el menú desplegable canal verde, y la selección de laMantenga imágenes fuente casilla de verificación. Analiza las películas a intervalos o instantáneas para determinar qué fagosomas se van a analizar. El canal de campo brillante es útil para esto. </li><li> Dibuje las regiones de interés (ROI) mediante la herramienta de selección poligonal óvalo alrededor fagosomas, y añadirlos a la gerente ROI haciendo clic en Análisis&gt; Herramientas&gt; Administrador ROI&gt; Agregar. Mida su relación de intensidad zimosán haciendo clic en Más&gt; Multi-Medida y de-seleccionar la una fila por cada rebanada de casilla. </li><li> Para las películas de lapso de tiempo, los fagosomas pista uno a la vez mediante la elaboración de un retorno de la inversión, tecleando Ctrl + M para medir cada punto de tiempo, y moviendo el retorno de la inversión cuando sea necesario para realizar un seguimiento de los valores de intensidad con el tiempo. Copie las mediciones de la ventana Resultados en una hoja de cálculo. Nota: El análisis de los 15 (5 cursos de tiempo por experimento x 3) experimentos independientes es ideal, pero más o menos puede ser necesaria en función de la variabilidad observada. Salvando las regiones de interés es siempre recomendable. Algunos paquetes de software permiten regi imagentración y actualización dinámica de rendimiento de la inversión, facilitando de esta parte del análisis. </li></ol></li><li> La conversión de fluorescencia para valores de pH <ol><li> Medir la relación 490/440 para fagosomas en las películas a intervalos de calibración como se describe en la sección 4.1. </li><li> En una hoja de cálculo, trazar los valores de la relación con el tiempo, y calcular los valores medios de relación de todos los fagosomas dentro del campo de visión (por lo general, entre 5-20) a partir de 5 puntos de tiempo dentro de la mitad del período de tiempo correspondiente a cada calibración pH solución. (Véanse también las cajas de color rojo en la figura 5A.) </li><li> Trazar estos medios 490/440 valores de la relación contra el pH medido real. Combine por lo menos 3 calibraciones independientes en un solo gráfico. Use un paquete de software estadístico o numérica para realizar una regresión no lineal (curva de mejor ajuste), por lo general a una ecuación sigmoidal. Nota: Por ejemplo, en la Figura 5B la ecuación utilizada fue una sigmoidal Boltzmann [Y = Bottom + ((Top-Bottom)/ (1 + EXP ((V50-X) / Slope))], donde los valores de Y son los 490/440 ratios, X son los valores de pH y la parte superior, inferior parámetros V50 y pendiente son calculados por el software. </li><li> Utilice la ecuación obtenida para transformar valores de la relación de pH. Nota: Por ejemplo, en la Figura 5B la ecuación obtenida para la curva de calibración en presencia de azida de sodio es 490/440 = 1,103 + Ratio [(2,89 a 1,103) / (1 ​​+ EXP ((6.993-pH) /0.9291)] . Resolviendo para pH da la siguiente ecuación: pH = 6.993 - 0,9291 * [ln ((1,178 / (Ratio-1.103)) - 1))]. </li></ol></li></ol>

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W., Geisow, M., Garcia, R., Harper, A., Miller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+respiratory+burst+of+phagocytic+cells+is+associated+with+a+rise+in+vacuolar+pH.">The respiratory burst of phagocytic cells is associated with a rise in vacuolar pH.</a> Nature. 290 (5805), 406-409 (1981).</li><li>Savina, 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=NOX2+controls+phagosomal+pH+to+regulate+antigen+processing+during+crosspresentation+by+dendritic+cells.">NOX2 controls phagosomal pH to regulate antigen processing during crosspresentation by dendritic cells.</a> Cell. 126 (1), 205-218 (2006).</li><li>Rybicka, J. M., Balce, D. R., Chaudhuri, S., Allan, E. R., Yates, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagosomal+proteolysis+in+dendritic+cells+is+modulated+by+NADPH+oxidase+in+a+pH-independent+manner.">Phagosomal proteolysis in dendritic cells is modulated by NADPH oxidase in a pH-independent manner.</a> EMBO J. 31 (4), 932-944 (2012).</li><li>Mantegazza, A. R., 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=NADPH+oxidase+controls+phagosomal+pH+and+antigen+cross-presentation+in+human+dendritic+cells.">NADPH oxidase controls phagosomal pH and antigen cross-presentation in human dendritic cells.</a> Blood. 112 (12), 4712-4722 (2008).</li><li>Balce, D. R., Allan, E. R., McKenna, N., Yates, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gamma-Interferon-inducible+lysosomal+thiol+reductase+(GILT)+maintains+phagosomal+proteolysis+in+alternatively+activated+macrophages.">gamma-Interferon-inducible lysosomal thiol reductase (GILT) maintains phagosomal proteolysis in alternatively activated macrophages.</a> J Biol Chem. 289 (46), 31891-31904 (2014).</li><li>Sanjurjo, L., 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=The+scavenger+protein+apoptosis+inhibitor+of+macrophages+(AIM)+potentiates+the+antimicrobial+response+against+Mycobacterium+tuberculosis+by+enhancing+autophagy.">The scavenger protein apoptosis inhibitor of macrophages (AIM) potentiates the antimicrobial response against Mycobacterium tuberculosis by enhancing autophagy.</a> PLoS One. 8 (11), e79670 (2013).</li><li>Yuan, Z., Zhi, L., Li-Juan, Z., Hong-Tao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Utility+of+Chloroquine+in+Cancer+Therapy.">The Utility of Chloroquine in Cancer Therapy.</a> Curr Med Res Opin. , 1-12 (2015).</li><li>Ohkuma, S., Poole, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+probe+measurement+of+the+intralysosomal+pH+in+living+cells+and+the+perturbation+of+pH+by+various+agents.">Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents.</a> Proc Natl Acad Sci U S A. 75 (7), 3327-3331 (1978).</li><li>El Chemaly, 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=VSOP/Hv1+proton+channels+sustain+calcium+entry,+neutrophil+migration,+and+superoxide+production+by+limiting+cell+depolarization+and+acidification.">VSOP/Hv1 proton channels sustain calcium entry, neutrophil migration, and superoxide production by limiting cell depolarization and acidification.</a> J Exp Med. 207 (1), 129-139 (2010).</li><li>Levine, A. P., Duchen, M. R., Segal, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+HVCN1+channel+conducts+protons+into+the+phagocytic+vacuole+of+neutrophils+to+produce+a+physiologically+alkaline+pH.">The HVCN1 channel conducts protons into the phagocytic vacuole of neutrophils to produce a physiologically alkaline pH.</a> bioRxiv. , (2014).</li><li>Al-Fageeh, M. B., Smales, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+and+regulation+of+the+cellular+responses+to+cold+shock:+the+responses+in+yeast+and+mammalian+systems.">Control and regulation of the cellular responses to cold shock: the responses in yeast and mammalian systems.</a> Biochem J. 397 (2), 247-259 (2006).</li><li>Yui, N., 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=Basolateral+targeting+and+microtubule-dependent+transcytosis+of+the+aquaporin-2+water+channel.">Basolateral targeting and microtubule-dependent transcytosis of the aquaporin-2 water channel.</a> Am J Physiol Cell Physiol. 304 (1), C38-C48 (2013).</li><li>Griffiths, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+phagosome+individuality+and+membrane+signalling+networks.">On phagosome individuality and membrane signalling networks.</a> Trends Cell Biol. 14 (7), 343-351 (2004).</li><li>Poburko, D., Santo-Domingo, J., 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=Dynamic+regulation+of+the+mitochondrial+proton+gradient+during+cytosolic+calcium+elevations.">Dynamic regulation of the mitochondrial proton gradient during cytosolic calcium elevations.</a> J Biol Chem. 286 (13), 11672-11684 (2011).</li></ol>

The authors have nothing to disclose.

Aunque más lento que los métodos alternativos, como la espectroscopia y FACS, que emplean una estrategia similar de uso de un colorante sensible al pH, junto a los objetivos, pero miden el pH promedio de una población de fagosomas, microscopía ofrece varias ventajas. En primer lugar es que interna y externa obligado, pero no interiorizada, las partículas pueden ser fácilmente distinguido sin tener que añadir otros productos químicos, tales como tripán azul o anticuerpos, para calmar o etiquetar partículas exte...

Phagocytosis, the process through which innate immune cells engulf large particles, evolved from the eating mechanism of single-celled organisms, and involves binding to a target, enveloping it with a membrane and pinching the membrane off to form a vacuole within the cytosol called a phagosome. While the phagosomal membrane is derived from the plasma membrane, active protein and lipid sorting, as well as fusion with endomembranes during phagosome formation, transform the phagosome into a distinct organelle within the cell with degradative properties that allow the killing, neutralization and breakdown of the ingested material1-3. This process, called phagosomal maturation, relies on the delivery of a host of proteolytic and microbicidal enzymes, including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase which transfers electrons into phagosomes producing the strong oxidant O2- and its derivative reactive oxygen species (ROS) 2,4. 
The luminal pH of phagosomes has a profound influence on several events required for phagosome function. First, pH influences trafficking of endomembranes in general, as pH-dependent conformational changes of transmembrane trafficking regulators alters the recruitment of trafficking determinants such as Arfs, Rabs and vesicular coat-proteins, which in turn define which vesicles may fuse with phagosomes 5-8. Second, the ionic composition of the phagosomal lumen is also transformed as phagosomes mature, and some ion transporters, such as the Na+/H+ exchanger or ClC family Cl-/H+ antiporters, which promote phagocytic function, rely on H+ translocation 9,10. Similarly, ROS production is intimately linked with phagosomal pH. ROS and its toxic oxidant byproducts have long been recognized as crucial for phagosomal killing in neutrophils 4,11,12, and have been shown to play critical roles in other phagocytes including macrophages, dendritic cells (DCs) and amoeba 13-16. The NADPH oxidase is an electrogenic enzyme that releases H+ in the cytosol as NADPH is consumed, and that requires the simultaneous transfer of H+ through companion HVCN1 channels alongside the transported electrons into the phagosomal lumen, in order to alleviate the massive depolarization that would otherwise lead to self-inhibition of the enzyme 17-21. Finally, several proteolytic enzymes have optimal activity at different pH, so time-dependent phagosomal pH changes can influence which enzymes are active and when. The importance of phagosomal pH is highlighted by organisms such as Mycobacterium tuberculosis, Franciscella tularensis and Salmonella typherium that subvert phagocytic killing at least in part by altering phagosomal pH 22-24.
In mammals the main phagocytes are neutrophils, macrophages and dendritic cells, and depending on cell type, time-dependent phagosomal pH changes can vary widely, and appear to play different roles. In macrophages a strong and rapid acidification mediated by the ATP-dependent proton pump vacuolar ATPase (V-ATPase) is one of the key factors mediating killing 25-27, resembling the mechanisms present in amoeba that use phagocytosis as an eating mechanism 28. In these cells activation of acidic proteases is thought to play a key role. In contrast, neutrophil killing relies more on ROS as well as HOCl produced by myeloperoxidase (MPO)11, and the pH remains neutral or alkaline during the first 30 min acidifying only later 29,30. Neutral pH has been suggested to favor the activity of oxidative proteases such as certain cathepsins. In DCs phagosomal pH is controversial, with some reporting acidification and others neutral or alkaline pH 31,32, but ROS and pH may profoundly influence the ability of these cells to present antigens to T cells, one of their main functions 33. 
Importantly, hormones, chemokines and cytokines may produce signaling events that induce maturation and changes in phagocyte behavior, and in turn influence phagosomal pH 34,35. Similarly, drugs, for example the antimalarial chloroquine, which is also considered for anti-cancer therapies 36, may affect phagosomal pH and therefore immune outcomes. Thus, a variety of cell biologists, immunologists, microbiologists and drug developers may be interested in measuring phagosomal pH when seeking to understand the mechanisms underlying the effects of a particular genetic disruption, bioactive compound or microorganism on innate and adaptive immune responses. 
Here we describe a method for measuring phagosomal pH in neutrophils that allowed us to show the importance of the HVCN1 channel in regulating neutrophil phagosomal pH 19. The method is based on the ratiometric property of fluorescein isothiocyanate (FITC) whose fluorescence emission at 535 nm is pH sensitive when excited at 490 nm but not 440 nm 37. When this dye is chemically coupled to a target, in this case zymosan, it can be followed using wide-field fluorescence microscopy, where cells are imaged as they phagocytose, and phagosomal pH changes are measured in real time as the phagosome matures. The actual pH is then gleaned by performing a calibration experiment where cells that have phagocytosed are exposed to solutions of different pH that contain the ionophores nigericin and monensin, that allow the rapid equilibration of the pH within phagosomes with the external solution. Ratio values are then compared to the known pH of solutions, a calibration curve is constructed by nonlinear regression and the resulting equation used to calculate pH from the ratio value.

<table>
	<tbody>
		<tr>
			<td>Zymosan A powder</td>
			<td>Sigma-Aldrich</td>
			<td>Z4250</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate</td>
			<td>Sigma-Aldrich</td>
			<td>F7250</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Anti-zymosan antibody (Zymosan A Bioparticles opsonizing reagent)</td>
			<td>Life Technologies</td>
			<td>Z2850</td>
			<td>Sigma-Aldrich O6637 is an equivalent product. Alternatively 25% serum can be used as an opsonizing reagent.</td>
		</tr>
		<tr>
			<td>4-Aminobenzoic hydrazide (4-ABH)</td>
			<td>Santa Cruz</td>
			<td>sc-204107</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Diphenyleneiodonium chloride (DPI)</td>
			<td>Sigma-Aldrich</td>
			<td>D2926</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Concanamycin A (ConcA)</td>
			<td>Sigma-Aldrich</td>
			<td>27689</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Nigericin</td>
			<td>Sigma-Aldrich</td>
			<td>N7143</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Monensin</td>
			<td>Enzo</td>
			<td>ALX-380-026-G001</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Life Technologies</td>
			<td>14200-075</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Hank&#39;s balance salt solution</td>
			<td>Life Technologies</td>
			<td>14025092</td>
			<td>Ringer&#39;s balanced salt solution or other clear physiological buffers may be substituted.</td>
		</tr>
		<tr>
			<td>Sodium carbonate (Na2CO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S7795</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>2-(N-Morpholino)ethanesulfonic acid (MES)</td>
			<td>Sigma-Aldrich</td>
			<td>M3671&#160;</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>N-Methyl-D-glucamine (NMDG)</td>
			<td>Sigma-Aldrich</td>
			<td>M2004&#160;</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid (EGTA)</td>
			<td>Sigma-Aldrich</td>
			<td>3777</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>&#160;Tris(hydroxymethyl)aminomethane (Tris)</td>
			<td>Sigma-Aldrich</td>
			<td>T1503&#160;</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Magnesium chloride (MgCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Absolute Ethanol (EtOH)</td>
			<td>Sigma-Aldrich</td>
			<td>2860</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Glass-bottom 35 mm petri dishes (Fluorodish)</td>
			<td>World Precision Instruments</td>
			<td>FD35-100</td>
			<td>Ibidi &#181;-clear dishes or coverslips with appropriate imaging chambers may be sustituted</td>
		</tr>
		<tr>
			<td>Sonicating water bath</td>
			<td>O. Kleiner AG</td>
			<td></td>
			<td>A sonicator may be used instead, various instrument providers exist</td>
		</tr>
		<tr>
			<td>Heamocytometer</td>
			<td>Marienfeld GmbH</td>
			<td></td>
			<td>Various instrument providers exist</td>
		</tr>
		<tr>
			<td>Widefield live imaging microscope</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td>Various instrument providers exist, but the microscope must be able to image 440/535 and 490/535 excitation/emission respective. Spinning disk confocal set-ups with brightfield capabilities may substituted, but zymosan tend to go out of focus more often.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Peristaltic pump (Dynamax RP-1)</td>
			<td>Rainin</td>
			<td></td>
			<td>Various instrument providers exist</td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Schott Gerate GmbH</td>
			<td></td>
			<td>Various instrument providers exist</td>
		</tr>
		<tr>
			<td>Manual Counter</td>
			<td>Milian SA</td>
			<td></td>
			<td>Various instrument providers exist</td>
		</tr>
	</tbody>
</table>

measuring phagosome ph by ratiometric fluorescence microscopy

La fagocitosis es un proceso fundamental a través del cual las células inmunitarias innatas engullen las bacterias, células apoptóticas u otras partículas extrañas con el fin de matar o neutralizar el material ingerido, o presentarlo como antígenos e iniciar la respuesta inmune adaptativa. El pH de los fagosomas es un parámetro crítico regulación de fisión o fusión con endomembranes y la activación de las enzimas proteolíticas, eventos que permiten la vacuola fagocítica a madurar en un orgánulo degradativa. Además, se requiere la translocación de H + para la producción de altos niveles de especies reactivas de oxígeno (ROS), que son esenciales para la matanza eficiente y señalización a otros tejidos del huésped. Muchos patógenos intracelulares subvierten asesinato fagocitosis mediante la limitación de la acidificación phagosomal, destacando la importancia del pH en la biología fagosoma. Aquí se describe un método radiométrico para la medición de pH phagosomal en los neutrófilos utilizando isotiocianato de fluoresceína (FITC) marcado con zymosan como targ fagocíticaets y imágenes de células vivas. El ensayo se basa en las propiedades de fluorescencia de FITC, que se inactivó mediante pH ácido cuando se excita a 490 nm pero no cuando se excita a 440 nm, lo que permite la cuantificación de una relación dependiente del pH, en vez de la fluorescencia absoluta, de un solo colorante. También se proporciona un protocolo detallado para llevar a cabo en la calibración de tinte situ y la conversión de la relación de a valores de pH real. Métodos radiométricos de un solo tinte se consideran en general superior a la longitud de onda única o protocolos pseudo-radiométrica de doble tinte, ya que son menos sensibles a las perturbaciones tales como blanqueo, se centran cambios, variaciones láser y etiquetado irregular, que distorsionan la señal medida. Este método se puede modificar fácilmente para medir el pH en otros tipos de células fagocíticas, y zymosan puede ser reemplazado por cualquier otra partícula que contiene amina, a partir de perlas inertes a los microorganismos vivos. Por último, este método puede ser adaptado para hacer uso de otras sondas fluorescentes sensibles a diferentes intervalos de pH o de otros phagosomactividades al, por lo que es un protocolo generalizado para la proyección de imagen funcional de fagosomas.

Los siguientes son resultados representativos de un experimento en el que el pH phagosomal de los neutrófilos primarios de ratón aislado de la médula ósea de tipo salvaje o Hvcn1 - / - los ratones fueron comparados. Para un experimento exitoso, es importante obtener suficientes fagosomas dentro del campo de visión durante toda la duración de la película de lapso de tiempo, evitando al mismo tiempo demasiados fagosomas, que más tarde serán más difíciles de segmento durante el an?...

Watch this Scientific Journal Video about Measuring Phagosome pH by Ratiometric Fluorescence Microscopy at JoVE.com

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

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.

Medición de pH Fagosoma por Ratiometric microscopía de fluorescencia

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

measuring-phagosome-ph-by-ratiometric-fluorescence-microscopy

Research

JoVE Journal

Immunology and Infection

13.9K vistas.  University of Geneva. 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. 

Video: Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

Asistida por microondas diagnóstico rápido de enfermedades de virus de plantas por microscopía electrónica de transmisión

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

Investigación

Inmunología e Infección

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

La visualización de las respuestas inducidas por toxinas bacterianas mediante microscopía de fluorescencia de células en vivo

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

Estudio de fagolisosoma Biogénesis en macrófagos en directo

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

La medición de las respuestas fisiológicas de<em&gt; Drosophila</em&gt; Las neuronas sensoriales a los lípidos feromonas Uso de Imagen de calcio en Vivo

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Ratiometric Imaging of Extracellular pH in Dental Biofilms

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 &#181;m thickness, but is limited to the pH range between 4.5 and 7.0.

A pH-sensitive ratiometric dye is used in combination with confocal laser scanning microscopy and digital image analysis to monitor extracellular pH in dental biofilms in real-time.

Imaging radiométrica de pH extracelular en los biofilms dentales

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites ...

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

&quot;Fagosoma Cierre de ensayo&quot; para visualizar Fagosoma Formación en tres dimensiones utilizando la reflexión interna total fluorescente Microscopía (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

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.

Imágenes de la Neutrófilos Fagosoma y el citoplasma usando un indicador de pH Ratiometric

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

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

Visualizar el lazo intracelular del tráfico de por vida de fluorescencia microscopía de imágenes

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of disease or occurrence of serious complications. Ektacytometry uses laser diffraction viscometry to measure the deformability of red blood cells subject to either increasing shear stress or an osmotic gradient at a constant value of applied shear stress. However, direct deformability measurements are difficult to interpret when measuring heterogenous blood that is characterized by the presence of both rigid and deformable red cells. This is due to the inability of rigid cells to properly align in response to shear stress and results in a distorted diffraction pattern marked by an exaggerated decrease in apparent deformability. Measurement of the degree of distortion provides an indicator of the heterogeneity of the erythrocytes in blood. In sickle cell anemia, this is correlated with the percentage of rigid cells, which reflects the hemoglobin concentration and hemoglobin composition of the erythrocytes. In addition to measuring deformability, osmotic gradient ektacytometry provides information about the osmotic fragility and hydration status of erythrocytes. These parameters also reflect the hemoglobin composition of red blood cells from sickle cell patients. Ektacytometry measures deformability in populations of red cells and does not, therefore, provide information on the deformability or mechanical properties of individual erythrocytes. Regardless, the goal of the techniques described herein is to provide a convenient and reliable method for measuring the deformability and cellular heterogeneity of blood. These techniques may be useful for monitoring temporal changes, as well as disease progression and response to therapeutic intervention in several disorders. Sickle cell anemia is one well-characterized example. Other potential disorders where measurements of red cell deformability and/or heterogeneity are of interest include blood storage, diabetes, Plasmodium infection, iron deficiency, and the hemolytic anemias due to membrane defects.

Here we present techniques to measure red cell deformability and cellular heterogeneity by ektacytometry. These techniques are applicable to general investigations of red cell deformability and specific investigations of blood diseases characterized by the presence of both rigid and deformable red cells in circulation, such as sickle cell anemia.

Medición de la deformabilidad y la heterogeneidad de glóbulos rojos en sangre por Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

In Vitro Formación de la trampa extracelular neutrófilo canino: Análisis dinámico y cuantitativo por microscopía de fluorescencia

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

Medición de pH Fagosoma por Ratiometric microscopía de fluorescencia

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