Damos las gracias a Steve Hansen McBryant y Jeff para ayudar con la ultracentrifugación y Kiser Patti para ayudar con el manejo del ratón. El Instituto Nacional de Enfermedades Neurológicas e Ictus de los Institutos Nacionales de Salud, otorgar 5R01NS056379-02 financiado este trabajo.

<html> 1. Purificación y Etiquetado de Prion Rods Este protocolo es una adaptación de otra publicada anteriormente 18 <ol><li> Homogeneizar 100 gramos de priones infectados con el tejido cerebral en 900 ml de hielo frío buffer de homogeneización (HB, 1X PBS que contenía 320 mM de sacarosa, NaCl 150 mM EDTA y 4 mM) 1 min a máxima velocidad en un comercial de la licuadora, luego 2 minutos en hielo. Repita tres veces. </li><li> Centrífuga homogeneizado durante 10 minutos a 3000 xg y 4 ° C. Retirar y guardar el sobrenadante en hielo. Volver a suspender el precipitado en 1 litro HB y repita los pasos 1.1 y 1.2. </li><li> Sobrenadantes piscina y centrifugar durante 60 minutos a 100.000 xg, 0 ° C. Desechar el sobrenadante. </li><li> Volver a suspender el precipitado en 100 ml de solución salina tamponada con Tris 1X (10 mM Tris-HCl, 0,1 mM de NaCl y 1 mM EDTA) que contiene 2% de Triton X-100 (TBST). Estimar la concentración de proteínas por el Bradford o ensayo similar y ajuste a 5 mg / ml en TBST. Se enfría en hielo durante 30 minutos. </li><li> Centrifugar durante 30 minutos a 100.000 xg, 0 ° C. Desechar el sobrenadante y lavar la pastilla con TBST 50 ml de 100 ml de TBS. Volver a suspender el precipitado en 100 ml de 1X PBS con 1% sarcosilo y cóctel de inhibidores de la proteasa y agitar durante 120 minutos a 37 ° C. </li><li> Capa de muestra en 900 ml de sacarosa 320 mM y se centrifuga durante 60 minutos a 100.000 xg, 10 ° C. Desechar el sobrenadante y volver a suspender el precipitado en 10 ml de NaCl 2,3 M, 5% sarcosilo. </li><li> Sonicar muestra de 5 x 40 segundos a máxima potencia un 70% en intervalos de 1 min en hielo. </li><li> Alícuota de 1 ml de muestra en tubos eppendorf y centrifugar durante quince minutos a 13.000 xg, 4 º C. Lávese las pastillas dos veces con TBS y almacenar @ -70 ° C o conjugado con fluorocromo en el paso 1.9. </li><li> Conjugado un tubo de agua purificada barras priones usando un vial de 649 DyLight fluorocromo de acuerdo con el protocolo del fabricante. </li><li> Intercambio DyLight buffer de etiquetado para PBS por diálisis o centrifugación a través de columnas de filtro. Almacenar en alícuotas de 20 l a -70 ° C, protegido de la luz hasta que esté listo para su uso. </li></ol> 2. La inoculación de priones y la recuperación de las células En esta sección se incluye la inoculación intraperitoneal de priones y la recuperación de las células del peritoneo, ganglios linfáticos del mediastino y mesentéricos y el bazo. <ol><li> Diluir las barras fluorescentes priones 1:50 en PBS inmediatamente antes del uso. Scruff ratón por la piel del cuello dorsal con el pulgar y el dedo índice, gire suavemente el ratón para que la cara y frenar la cola con el dedo meñique. Supinación del ratón, elevando el torso traseras por encima de la cabeza y sosteniéndolo un poco al revés. </li><li> Con una jeringa de insulina 30G, se inyectan 100 l de las barras de priones fluorescente 1 cm por fuera de la línea media y de 1-2 cm por delante de la articulación sacroilíaca. Insertar la aguja 1 cm a través de la piel dirigido a 45 ° en sentido medial. Utilice dilución 1:50 de perlas fluorescentes o solo PBS como control. Incubar los ratones de tiempo asignado antes del análisis. </li><li> La eutanasia a los ratones de acuerdo con el cuidado de animales aprobada con el protocolo del comité. Inyectar 10 ml de PBS en la cavidad peritoneal con una jeringa de 12 mL y una aguja 25G. Agite suavemente la canal longitudinal durante un minuto. Recuperar la suspensión de células de peritoneo con la misma jeringa y una aguja 16G en un tubo cónico de 15 ml y reservar en el hielo, mientras que la disección de cadáveres continúa. </li><li> Humedezca la parte ventral del cuerpo a fondo con etanol al 70%. Levante la piel en la línea media justo por debajo de las costillas con las pinzas y cortar una pequeña incisión a través de la piel con unas tijeras. A partir de allí, hacer una incisión de 3-5 cm de largo a través de la piel de en ambas direcciones a la cabeza y la cola, y después dos incisiones de 2 cm de la línea media lateral de cada lado. Pele y el pin de la piel para revelar las cavidades peritoneal y torácica. Corte cuidadosamente a través de las delgadas membranas que rodean el peritoneo transparente y reflejar el tórax para revelar el mediastino. </li><li> Encontrar y eliminar 2-4 ganglios linfáticos del mediastino, ligeramente dorsal y lateral adyacente a la timo, medial adyacente a la arteria innominada, cerca de la cara ventral de la tráquea. Encontrar y eliminar un máximo de seis ganglios linfáticos mesentéricos, integrado en el tejido mesentérico blanca y suave que las anclas de los intestinos a la pared posterior del abdomen. Encontrar y extirpar el bazo, un órgano largo, de color rojo oscuro situada justo lateral de la línea media dorsal y en los intestinos en el lado izquierdo del abdomen. Ganglios linfáticos de la Reserva en 1 ml de medio RPMI 1640 en hielo hasta que la disección se ha completado. </li><li> Macerar y pulse ganglios linfáticos a través de un colador de malla de 40 micras en 3 ml de RPMI en una placa Petri de plástico con el émbolo de una jeringa de 1 ml. Enjuague colador con un adicional de 2 ml de RPMI y la transferencia de la suspensión de 5 ml de células a un tubo cónico de 15 ml. Enjuague la placa de Petri con un adicional de 5 ml de RPMI y traslado al mismo tubo. Centrifugar 5 min a 250 xg, desechar el sobrenadante, resuspender el pellet de células en 1 ml de tampón FACS (PBS 1X, 1% albúmina de suero bovino y 10 mM EDTA) y transferir a un tubo Eppendorf de 1,5 ml. </li></ol><html> ove_title &quot;&gt; 3. tinción celular y citometría de flujo El siguiente es un protocolo de tinción típica para la identificación de subgrupos de células inmunes con anticuerpos fluorescentes contra el bien caracterizado marcadores inmunológicos de superficie celular. Todos los anticuerpos se diluyeron en tampón FACS inmediatamente antes del uso. <ol><li> Recuento de células y alícuota de 10 6 células en tubos Eppendorf. Las células se centrifuga (véase 2.7) y lavar la pastilla 2 veces con 1 ml de tampón FACS. </li><li> Se incuban las células 20 minutos sobre el hielo en 100 L de 2 ng / ml rata anti-ratón CD16/32 para bloquear los receptores Fc endógeno. Pellet y las células de lavado con tampón FACS. </li><li> Añadir 100 ml de 1 ng / mL anticuerpos fluorescentes adecuadas (y) s para cada muestra e incubar en hielo durante una hora. Añadir 100 ml de solución tampón FACS sólo a las células control. Pellet y las células se lavan dos veces con tampón FACS. </li><li> Vuelva a suspender las células en 1 ml de tampón ACK (150 mM NH 4 Cl, 1 mM KHCO3 y 0,1 mM EDTA) Incubar en hielo durante 1 min para lisar los glóbulos rojos. Pellet y las células de lavado con tampón FACS. Vuelva a suspender las células en 1 ml de tampón FACS. </li><li> Recopilar datos a partir de células con un citómetro de flujo capaz de detectar multicolor y análisis, por lo general con láser 405, 488 y 633 nm de excitación y cinco a diez detectores de fluorescencia. </li></ol> 4. Los resultados representativos: Nos purifica barras priones de crudo, los priones infectados homogenato de cerebro (Figura 1) solubilización detergente y ultracentrifugación de la E2 alces cerebro homogeneizado (calles 1 y 2) enriquecido para las barras de priones (comparar los carriles 1 y 3), que fue también la proteasa K resistentes (calle 4). Curiosamente, sin digerir purificada barras priones muestran un perfil muy similar a glicoformas homogeneizado digerido cerebro E2, lo que indica el enriquecimiento espectacular de los priones. Homogeneizado de idéntico tratamiento normal del cerebro, no produjo ningún PK-resistentes PrP C (carriles 5 y 6). Análisis de citometría de flujo de las poblaciones de células específicas de demostrar que las células presentadoras de antígenos de origen hasta el peritoneo, por dos horas y mantener cuentas fluorescentes y las barras de priones, como se evidencia por fluorescencia aumentado dramáticamente en los controles de PBS inyectado (Figura 2). No hay células recolectadas del peritoneo de PBS controles tratados muestran fluorescencia por encima del fondo (Fig. 2A-G), mientras que las células de cordón (Fig. 2H-N) y priones (Figs. 2O-U)-inocularon ratones muestran fluorescencia significativo, especialmente en los monocitos (Fig. 2I y P), las células dendríticas (Fig. 2K y R) y los macrófagos (Fig. 2L y S), y algunos neutrófilos (Figura 2J y Q) y el menor número de células B (2T Figura). Bolas y teniendo priones monocitos, células dendríticas y los macrófagos también se encuentran en los ganglios linfáticos del mediastino dos horas después de la inoculación (Fig. 2AD y AK, AF y AG y AM y AN, respectivamente), mientras que pocos o ningún PrP CWD-teniendo neutrófilos , las células B o T se encontraron allí (Figs. 2AE y AL, AH y AG y AI y AP, respectivamente). No se detectaron granos o barras de priones en el bazo o los ganglios linfáticos mesentéricos (datos no mostrados). En su totalidad, estos datos demuestran que el tráfico de las células inmunes priones de manera muy similar a los antígenos de partículas. <table border="1"><tbody><tr><td> REACTIVO </td><td> Fluorocromo </td><td> Excitación láser (nm) </td><td> Pico de emisión (nm) </td></tr><tr><td> Barras de priones </td><td> DyLight 649 </td><td> 633 </td><td> 674 </td></tr><tr><td> 1 m cuentas </td><td> AlexaFluor 660 </td><td> 633 </td><td> 685 </td></tr><tr><td> Anticuerpos </td><td></td><td></td><td></td></tr><tr><td> CD11b </td><td> eFluor 450 </td><td> 405 </td><td> 450 </td></tr><tr><td></td><td> Ficoeritrina Cy7 </td><td> 488 </td><td> 760 </td></tr><tr><td> CD11c </td><td> R-ficoeritrina </td><td> 488 </td><td> 575 </td></tr><tr><td></td><td> Ficoeritrina Cy7 </td><td> 488 </td><td> 760 </td></tr><tr><td> Ly6C </td><td> Fluoroisothiocyante </td><td> 488 </td><td> 518 </td></tr><tr><td> Ly6G </td><td> R-ficoeritrina </td><td> 488 </td><td> 575 </td></tr><tr><td> CD21 </td><td> DyLight 488 </td><td> 488 </td><td> 518 </td></tr><tr><td> B220 </td><td> Aloficocianina-Cy7 </td><td> 633 </td><td> 785 </td></tr><tr><td> CD3 </td><td> R-ficoeritrina </td><td> 488 </td><td> 575 </td></tr></tbody></table>&quot;&gt; La Tabla 1. Propiedades espectrales de los reactivos fluorescentes <img src="/files/ftp_upload/2349/2349fig1.jpg" alt="Figura 1" /> Figura 1. Purificación de las barras de priones de homogenato de cerebro infectado. Se utilizó un homogeneizado de cerebro crudo de 10% a partir de un ciervo CWD-infectados (E2, carriles 1 y 2) como material de partida desde el que purifica las barras agregados de priones (carriles 3 y 4). Crudo y los materiales purificados mostró resistencia característica de la proteasa K tratamiento (calles 1 - 4), mientras que homogenato de cerebro normal no (carriles 5 y 6). Marcadores de peso molecular se muestran en la Kd a la izquierda de la mancha. BH, homogeneizado de cerebro. <img src="/files/ftp_upload/2349/2349fig2.jpg" alt="Figura 2" /> Figura 2. Análisis de citometría de flujo de tráfico de las células inmunes priones del peritoneo a los ganglios linfáticos del mediastino dos horas después de la inoculación. PBS (paneles AG y AB-V), perlas fluorescentes (HN y AC-AI) o las barras de priones (OU y AJ-AP) se se inyecta en el peritoneo de los ratones y las células recolectadas a partir del líquido peritoneal lavado (UA) o los ganglios linfáticos del mediastino (V-AP) dos horas después. Los gráficos en las células muestran primero la columna de los ratones tratados con PBS (paneles A y V), perlas fluorescentes (panel H y AC) y las barras de priones (paneles O y AJ). Células fluorescentes (puntos de color rojo o verde) y el total de células (puntos grises) se grafican para mostrar el tamaño relativo (de dispersión hacia adelante), granularidad (dispersión lateral) y la proporción del total de células vivas que presentan fluorescencia. Un número significativo de células características de los granulocitos retenido perlas fluorescentes y las barras. Las células se tiñeron también con anticuerpos contra inmune marcadores de superficie celular y cerrado para AVG-Ly6C + monocitos (paneles B, I, P, W, AD y AK), Ly6c-CD11c-CD11b + Ly6G + neutrófilos (C, J, Q, X, AE y AL), Ly6G-Ly6C-CD11b + CD11c + en las células dendríticas (D, K, R, Y, AF y AM), Ly6G-Ly6C-CD11c-CD11b + macrófagos, (E, L, S, Z AG, y AN), CD3 -B220 + CD21 + células B (F, M, T, AA, AH y AO) y CD21-B220-CD3 + células T (G, N, U, AB, AI y AP). Monocitos, células dendríticas y los macrófagos conservado perlas fluorescentes y las barras de priones en la cavidad peritoneal (Grupos I y P, K y R y L y S, respectivamente) y las transportó a los ganglios linfáticos del mediastino (paneles AD y AK, AF y AG y AM y AN, respectivamente), lo que demuestra la absorción similares y el tráfico de estas dos partículas. Neutrófilos retenido una cantidad significativa de granos (J) y menos barras (Q), pero no para entregarlos a los ganglios linfáticos (AE y AL). Las células B retenido y transportado unas varillas (T y AO), pero prácticamente no hay cuentas (M y H). Las células T de tráfico ni perlas, ni barras (N, U, AI y AP).

<ol>
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Aquí se demuestra un protocolo para el etiquetado y el seguimiento de los priones en vivo que facilita enormemente el control los primeros eventos en la infección por priones periféricos. Este protocolo mejora en gran medida en anteriores intentos de control de la utilización de priones in vitro e in vivo 9 3,12 por pre-etiquetado inóculo priones altamente enriquecido de forma inequívoca la diferencian de PrP C endógena. Debido a que los priones infecciosos y ...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>CWD-infected elk brain</td>
<td>&nbsp;</td>
<td>Private elk farm in Colorado</td>
<td>&nbsp;</td>
<td>Use any non-human prion-infected brain</td>
</tr>
<tr>
<td>Blender</td>
<td>&nbsp;</td>
<td>Oster</td>
<td>6694-015</td>
<td>Use any commercial blender</td>
</tr>
<tr>
<td>Centrifuge</td>
<td>&nbsp;</td>
<td>Sorvall</td>
<td>SS34 rotor</td>
<td>Use any centrifuge /rotor that can reach 3000 x g and hold &ge; 500 ml volumes</td>
</tr>
<tr>
<td>Ultracentrifuge</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>50.2 Ti rotor</td>
<td>Use any ultracentrifuge /rotor that can reach 100,000 x g and hold &ge; 500 ml volumes</td>
</tr>
<tr>
<td>Bradford Reagent</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>B6916</td>
<td>&nbsp;</td>
<tr>
<td>Complete mini protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11 836 170 001</td>
<td>&nbsp;</td>
<tr>
<td>Sonicator</td>
<td>&nbsp;</td>
<td>Misonix</td>
<td>MP4000X</td>
<td>Use any horn or probe sonicator set to ~70% max power</td>
</tr>
<tr>
<td>DyLight antibody Labeling kit</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>53050</td>
<td>&nbsp;</td>
<tr>
<td>microcentrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>55430R</td>
<td>Use any refrigerated microcentrifuge that can achieve 13,000x g</td>
</tr>
<tr>
<td>centrifugal filter columns</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>Microcon YM-100</td>
<td>Use any filter or dialysis membrane with 100 Kd molecular weight cutoff</td>
</tr>
<tr>
<td>8-40 week-old FVB mice</td>
<td>&nbsp;</td>
<td>Charles River</td>
<td>207</td>
<td>Use any inbred mouse strain</td>
</tr>
<tr>
<td>1 &mu;m red fluorescent beads</td>
<td>&nbsp;</td>
<td>Phosphorex</td>
<td>2307</td>
<td>Use any fluorescent bead &le; 10 &mu;m</td>
</tr>
<tr>
<td>RPMI 1640 medium </td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11875-093</td>
<td>&nbsp;</td>
<tr>
<td>40 &mu;m cell strainer</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352340</td>
<td>&nbsp;</td>
<tr>
<td>fluorescent antibodies</td>
<td>&nbsp;</td>
<td>BD pharmingen</td>
<td>Various</td>
<td>Use any fluorescent antibody appropriate for your application.</td>
</tr>
<tr>
<td>flow cytometer</td>
<td>&nbsp;</td>
<td>Dakocytomation</td>
<td>CyanADP</td>
<td>Use any flow cytometer capable of multicolor fluorescence detection</td>
</tr>		</tbody>
		</table>
		</div>

monitoring immune cells trafficking fluorescent prion rods hours after intraperitoneal infection

Presencia de una forma anormal de una proteína priónica codificada por el hospedador (PrP), que es resistente a la proteasa, patológicos e infecciosos caracteriza a las enfermedades por priones, tales como caquexia crónica (CWD) de los cérvidos y la tembladera en las ovejas. La hipótesis del prión afirma que esta conformación anormal constituye la mayor parte o la totalidad de los priones infecciosos. El papel del sistema inmunológico en los primeros eventos en la patogénesis del prión periférico ha sido demostrado de manera convincente de la caquexia crónica y 1-3 scrapie. Transgénicos y los estudios farmacológicos en ratones revelaron un papel importante del sistema del complemento en la retención y la reproducción de los priones poco después de la infección por 4-6. In vitro e in vivo también han observado la retención de priones por las células dendríticas 70-10, aunque su papel en el tráfico sigue siendo claro 11-16. Los macrófagos también han sido implicados en la patogénesis del prión temprano, pero estos estudios se han centrado en los acontecimientos que ocurren semanas después de la infección 3,11,17. Estos estudios previos también sufren el problema de diferenciar entre endógeno PrP C y los priones infecciosos. Aquí se describe un método semicuantitativo, imparcial para evaluar la absorción de priones y el tráfico del sitio de la inoculación de las células inmunes reclutados en ella. Barras de agregados de priones fueron purificados de homogenato de cerebro infectado por solubilización de detergente no agregados y proteínas ultracentrifugación a través de un colchón de sacarosa. Electroforesis en gel de poliacrilamida, teñidos de azul Coomassie y el oeste de la recuperación de las barras de Blot confirmó priones altamente enriquecido en la fracción de gránulos. Barras de priones por vía intraperitoneal fueron marcados con fluorocromos luego inyectadas en ratones. Dos horas más tarde, las células inmunes del líquido lavado peritoneal, el bazo y los ganglios linfáticos del mediastino y mesentéricos se analizaron para la retención de priones vara y subconjuntos de células identificadas mediante citometría de flujo multicolor con marcadores de monocitos, neutrófilos, células dendríticas, macrófagos y células B y T. Este ensayo permite el control directo por primera vez de las células inmunes y la adquisición de los priones en el tráfico vivo en cuestión de horas después de la infección. Este ensayo también se diferencia claramente los priones infecciosos, agregados de PrP normalmente se expresa en las células huésped, que puede ser difícil y llevar a problemas de interpretación de los datos en los sistemas de ensayo de otros. Este protocolo se puede adaptar a las rutas de inoculación otros (oral, intranervous intravenosa y subcutánea, por ejemplo) y los antígenos (cuentas conjugado, los patógenos bacterianos, virales y parasitarias y las proteínas, el huevo), así.

Watch this Scientific Journal Video about Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection at JoVE.com

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

A continuación se describe un ensayo de novela de Vigilancia de la aceptación de priones y el tráfico por las células inmunes inmediatamente después de la inoculación intraperitoneal de purificación y el etiquetado fluorescente barras de agregados de priones a partir de material cerebral infectado entonces el seguimiento de su consumo y el movimiento del punto de inyección y la caracterización de las células mediadoras de estos eventos.

Monitoreo células inmunes Tráfico Varillas fluorescente priones horas de la infección intraperitoneal

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such ...

monitoring-immune-cells-trafficking-fluorescent-prion-rods-hours

Research

JoVE Journal

Immunology and Infection

11.4K vistas.  Colorado State University. A continuación se describe un ensayo de novela de Vigilancia de la aceptación de priones y el tráfico por las células inmunes inmediatamente después de la inoculación intraperitoneal de purificación y el etiquetado fluorescente barras de agregados de priones a partir de material cerebral infectado entonces el seguimiento de su consumo y el movimiento del punto de inyección y la caracterización de las células mediadoras de estos eventos.

Video: Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Medición de la actividad calpaína en células fijadas y vivir por citometría de flujo

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Investigación

Inmunología e Infección

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Siete pasos para células estrelladas

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Análisis por citometría de flujo de células inmunes dentro de aortas murino

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Aislamiento de células inmunes funcionales cardíacos

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

A raíz de la celda de destino en<em&gt; E. coli</em&gt; Después de la infección por fagos Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Citometría de flujo El aislamiento de células primarias murino tipo II alveolares epiteliales de Estudios funcionales y moleculares

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

Aislamiento de tejido adiposo células inmunes

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

Generación de nódulos linfáticos de grasa Pad quimeras para el Estudio del Ganglio Linfático estroma origen de célula

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

La detección de nanopartículas fluorescentes Interacciones con inmunodeficiencia primaria subpoblaciones celulares por citometría de flujo

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

Un enfoque semi-automatizado de preparación de cócteles de anticuerpos para el Análisis Inmunofenotípicos de la sangre periférica humana

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...