Damos las gracias a Aaron Rae (Universidad de Emory, Departamento de Pediatría y Salud Infantil de núcleo de flujo de Atlanta) para la clasificación de células. Este trabajo fue apoyado por el NIH subvención AA01787001, el Premio de Desarrollo de Carrera de la enfermedad de Crohn y Colitis Foundation of America, y una investigación de Emory-Egleston Children Center de semillas de subvención para TLD

1. La disección y la disociación de las células intestinales Preparación de los reactivos y equipos: <ul><li> Cálido Ca 2 + / Mg 2 + libre de PBS (CMF PBS) a temperatura ambiente. </li><li> Caliente Ca 2 + / Mg 2 +-libres HBSS con FBS al 5% (HBSS CMF / FBS) y 2 mM EDTA a temperatura ambiente. </li><li> Cálido agitador orbital a 37 º C. </li></ul> Nota: Los pasos de 1,1 a 1,7 debe realizarse lo más rápidamente posible para minimizar el grado de muerte celular y para lograr el máximo rendimiento celular. <ol><li> La eutanasia de los ratones en una cámara de CO 2 y etanol al 70% de pulverización en el abdomen y el tórax. </li><li> Hacer una pequeña incisión horizontal en el centro del abdomen con una tijera y remueva la piel para exponer el peritoneo. </li><li> Proceder a separar el estómago en el intestino delgado superior por el corte en el esfínter pilórico. Tease de distancia del mesenterio con unas pinzas y cortar de nuevo enla válvula ileocecal para liberar todo el intestino delgado del intestino grueso. Haga un corte en el orificio anal y otra vez separar el mesenterio del intestino grueso hasta que esté libre. </li><li> Cortar los dos puntos longitudinalmente con tijeras y lavar contenido fecal y el moco de la luz intestinal en CMF PBS a temperatura ambiente. </li><li> Con unas tijeras y pinzas de disección macroscópica de las placas de Peyer a lo largo de la superficie antimesentérico del intestino delgado y se abre el intestino delgado en sentido longitudinal. </li><li> Lavar el lumen del intestino delgado de contenido fecal y moco en CMF PBS a temperatura ambiente. </li><li> Por otro corte en el intestino delgado / de gran tamaño en aproximadamente 1,5 cm de piezas y ponerlas en diferentes tubos de 50 ml cónicos que contienen 30 ml de pre-calentado HBSS CMF / FBS y EDTA 2 mM. No agregue más de un intestino por 50 ml tubo cónico. </li><li> Horizontalmente colocar cada tubo cónico de 50 ml en un agitador orbital y agitar durante a 250 rpm durante 20 min a 37 ° C. &lt;/ Li&gt; <li> Colocación de un filtro de malla de alambre sobre un solo cubo de la basura y verter el contenido de cada tubo cónico de 50 ml a través de recuperar las piezas de 1,5 cm de intestino y colocarlos en diferentes tubos de 50 ml cónicos que contienen 30 ml de precalentado CHBSS / FBs con 2 mM EDTA. </li><li> Repita 1.8. </li></ol> 2. La digestión del tejido y aislamiento de las células intestinales Preparaciones de reactivos y equipamiento: <ul><li> Colagenasa solución: 1,5 mg / ml de colagenasa tipo VIII disolvió en pre-calentado HBSS CMF / FBS con 40 mg / ml de DNasa I. </li><li> Pre-caliente CMF HBSS / FBS y EDTA 2 mM. </li><li> Pre-caliente agitador orbital a 37 º C. </li><li> Helada CMF HBSS / SFB. </li></ul><ol><li> Después de la segunda ronda de agitación, verter el contenido de cada tubo cónico de 50 ml a través del filtro y transferir 1,5 cm piezas de intestino a un pequeño de plástico pesan barco después frotando suavemente el exceso de material de distancia usando una toalla de papel. </li><li> Rápidamente el picadillo de un.5 Piezas cm de intestino usando tijeras directamente en el bote de peso y añadir intestino picada a 20 ml de solución de colagenasa. Horizontalmente colocar cada tubo cónico de 50 ml en un agitador orbital y digerir a 200 rpm durante 10-20 min a 37 ° C. Por favor, ver más adelante en relación con la optimización. </li><li> Brevemente vórtice para asegurar la disociación completa de cualquier tejido restante intestinal y filtrar a través de un colador micras 100 células directamente en un tubo cónico de 50 ml. </li><li> Parte superior de cada tubo cónico de 50 ml con HBSS CMF / SFB y se centrifuga a 1500 rpm durante 5 min a 4 ° C. Si un sólido precipitado no se observa para las muestras de colon después de la centrifugación, las muestras deben ser centrifugadas de nuevo por 3,5 min. Repita este paso de lavado, una vez más. </li><li> Retirar el sobrenadante y resuspender el botón celular en el hielo frío HBSS CMF / SFB y muestras lugar en el hielo. </li><li> Proceda a la Sección 3 para la adquisición de FACS / o análisis de la Sección 4 de cordón magnético de enriquecimiento para celular sorti de alta velocidadng. </li></ol> 3. Anticuerpos de tinción para el Análisis de Flujo Multi-Color de citometría de países en desarrollo y los macrófagos Preparaciones de reactivos y equipamiento: <ul><li> Helado de PBS CMF. </li><li> Helado de buffer de tinción (CMF PBS + 5% de SFB). </li><li> Prepare la mancha de células muertas en el hielo frío CMF PBS a una dilución 1:1000 con LIVE / DEAD Se fija Kit de Aqua Dead Cell Mancha. </li><li> Preparar el cóctel tinción de anticuerpos mediante la adición de los siguientes fluorescencia-etiquetados anticuerpos monoclonales (mAb) a la tampón enfriado con hielo tinción: CD45-PerCP, PE-CD103, CD11c APC-, MHC II-(IA b)-Alexa Fluor 700, CD11b -eFluor 450, F4/80-PE-Cy7. </li></ul><ol><li> Transferir las células en un 5 poliestireno ml de fondo redondo (FACS) del tubo. </li><li> Lave las células dos veces en helado CMF PBS. </li><li> Incube las muestras con células muertas mancha durante 15 minutos en hielo en la oscuridad. </li><li> Lave las células dos veces en helado CMF PBS. </li><li> Las células de bloques con 2.4G2 anti-FcγRIII/II en helado tincióntampón durante 10 minutos en hielo. </li><li> Lavar las células en tampón de tinción enfriado con hielo. </li><li> Incubar las muestras con cóctel de anticuerpos tinción durante 20 minutos en hielo en la oscuridad. </li><li> Lavar las células con tampón enfriado con hielo tinción dos veces y resuspender las muestras en 400 l de tampón enfriado con hielo tinción y pasar a través de la tapa 40 micras filtro en tubos FACS. </li><li> Adquirir muestras de LSR II citómetro (BD), según la definición de la estrategia de puerta en la Sección 5 y la Figura 1. </li></ol> 4. El enriquecimiento de los países en desarrollo y los macrófagos en el intestino Preparaciones de reactivos y equipamiento: <ul><li> Helado de buffer de tinción (CMF PBS + 5% de SFB). </li></ul><ol><li> Incubar única suspensión celular obtenida de la etapa con 2,5 CD11b y perlas MACS CD11c de acuerdo con las instrucciones del fabricante. </li><li> Lavar las células con tampón de tinción enfriada con hielo, seguido por centrifugación. </li><li> Desechar el sobrenadante y resuspender el sedimento celular en un tampón de tinción ml enfriado con hieloy pasar a través de un colador micras 100 celular, seguido por un filtro de células 40 micras. </li><li> Enriquecer de bolas magnéticas adheridas a las células por la selección positiva utilizando MACS columna LS magnético. </li><li> Repita el paso 4.2 y desechar el sobrenadante. </li><li> Se incuban las células con mAbs marcador de superficie tal como se describe en el paso 3,7. </li><li> Lavar magnéticos talón enriquecidos con células dos veces con tampón de tinción enfriado con hielo. Resuspender sedimentos celulares en tampón de 500 l tinción helado sin azida de sodio, y pasan a través de 40 micras filtro de células en un tubo FACS. </li><li> Proceda a la FACS, la clasificación en el Clasificador de BD ARIA de la célula para ordenar II DC intestinal y / o subconjuntos de macrófagos de interés. </li></ol> 5. Estrategia para la apertura de puerta LP APC Nota: Obsérvese que no teñidas células intestinales se puede utilizar como un control negativo para ayudar en la colocación correcta de las puertas para separar poblaciones positivas y negativas. <ol><li> Como se muestra en la Figura1, crear un diagrama de puntos y no incluyen células que son positivas para la tinción de células muertas (fig. 1A), seguido por la exclusión de eventos doblete (Fig. 1B y C). Entonces, la puerta de las células de interés de acuerdo para avanzar y dispersión lateral asegurarse de excluir a los residuos (Fig. 1D). </li><li> Crea otro gráfico de puntos y la puerta sobre CD45 + y b + IA células, lo que caracteriza fenotípicamente APC (Fig. 1E). </li><li> En un gráfico de puntos por separado, el análisis de la expresión de CD11b y CD11c para distinguir las DC específico y subconjuntos de macrófagos (R1, R2 y R3;. Fig. 1F). Las células de R1 son CD11c + CD11b mate / - las células. La región, R2, se delinea de manera que estas células tienen niveles similares de expresión en la superficie para CD11c como en las células de R1 pero las células de R2 también CD11b expresa. Después de ello, la región R3 es designado para las células que son + CD11b y opaco / CD11c -. </li><li> Uncatalizar regiones R1, R2, R3 y de expresión adicional para F4/80 y CD103 para diferenciar los macrófagos y las poblaciones de CC, respectivamente. El CD11c + CD11b aburrida / - las células de R1 expresan niveles elevados de la integrina αE, CD103, y bajos niveles de F4/80 (Fig. 1G). CD11b + CD11c + en las células de R2 se componen de los países en desarrollo y los macrófagos sobre la base de su expresión dicotómica de CD103 y F4/80 (Fig. 1H). Por último, CD11b + CD11c mate / - las células de los macrófagos constituyen R3 en función del perfil fenotípico de F4/80 + y CD103 - (Fig. 1i) 16. </li></ol> 6. Los resultados representativos <img alt="Figura 1" src="/files/ftp_upload/4040/4040fig1.jpg" /> Figura 1. La estrategia de apertura de puerta para los países en desarrollo y los macrófagos intestinales. Las células muertas (A) y dobletes (B y C) fueron excluidos del análisis primero y luego pequeños intestinalnales células fueron separadas de acuerdo a la dispersión frontal y lateral (D), y vehículos blindados se define como CD45 + IA + B (E). Los macrófagos y los DC se identificaron mediante la expresión de CD11b y CD11c (F). CD103 y F4/80 expresión para células pre-bloqueado en R1 (G), R2 (H) y R3 (I) se analizó la población. <img alt="Figura 2" src="/files/ftp_upload/4040/4040fig2.jpg" /> Figura 2. Rendimiento de la célula y la calidad tinción de anticuerpos depende del tiempo de digestión. CD11b y CD11c patrón de tinción y el rendimiento total de células de bajo-(A, D), de manera óptima-(B, E) o más digerido-(C, F) del tejido intestinal. Células intestinales fueron aisladas de un intestino ratón C57BL / 6 pequeño y los DC y los macrófagos se analizaron por FACS en el II BD LSR. Voltaje y la indemnización se establece mediante teñidas y de un solo fluorocromo manchadas de esplenocitos. Las células muertas (Fig. 1A) y dobletes (Fig. 1B y C) fueron excluidos de primerael análisis. Las células de interés se analizaron a continuación de acuerdo con frontal y lateral de dispersión (Fig. 1D), seguido por inyección en CD45 + b + e IA células (Fig. 1E). Posteriormente, CD11b y CD11c expresión se evaluó entre el CD45 + IA b + células para delinear tres regiones (R1, R2 y R3;. Fig. 1F). CD103 y F4/80 expresión en las tres regiones se evaluó para distinguir entre los DC y macrófagos, respectivamente. El CD11c + CD11b aburrida / - las células de R1 expresaron altos niveles de la integrina αE, CD103, y bajos niveles de F4/80 (Fig. 1G). CD11b + CD11c + en las células de R2 se compone de los dos países en desarrollo y los macrófagos sobre la base de su expresión dicotómica de CD103 y F4/80 (Fig. 1H), mientras que CD11b + CD11c mate / - las células de R3 constituyen los macrófagos basados ​​en el perfil fenotípico de F4 / 80 + y CD103 - ( <strong&gt; fig. 1I) 16. Los macrófagos dentro de la puerta de R2 y los macrófagos en la puerta de R3 tiene de avance similar y las propiedades de dispersión y secundarios se distinguen por la expresión de CD11c. La dicotomía funcional de estos subconjuntos sigue siendo no del todo comprendidas. La relación entre la duración de la digestión del tejido sobre el rendimiento total de células y la expresión de CD11b y CD11c se ilustra en la Figura 2. Tejido intestinal que se digirió durante 3 minutos (bajo-digestión) produjo bajo número total de células (Fig. 2D) y por lo tanto pocos países en desarrollo y macrófagos disponibles para la caracterización (Fig. 2A). Digestión de tejido durante 11 minutos produce un rendimiento sólido de células vivas (Fig. 2E) con poblaciones de países en desarrollo y los macrófagos que expresaban altos niveles de CD11b y CD11c y eran fenotípicamente distinta (Fig. 2C). En contraste, la digestión durante 50 min (sobre-digestión) resultó en un rendimiento celular similar cuando compared a la digestión optimizada (Fig. 2E y F), sin embargo, la delineación de diferentes poblaciones de células que utilizan CD11b y CD11c se hizo más oscuro como la expresión de CD11c disminuida (Fig. 2C) y el número de células muertas se incrementó (datos no presentados).

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<img src="/files/ftp_upload/4040/4040fig3.jpg" alt="Figura 3"/> Figura 3. Factores importantes para la optimización del rendimiento de la célula y la expresión del antígeno de superficie. Rendimiento de la célula y la expresión del antígeno de superficie son afectados directamente por la duración de la digestión del tejido, las características específicas de la colagenasa, el grado de tejido picado, y la presencia o ausencia de inflamación, que puede afectar a la integridad del tejido y...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> PBS 1X, Ca 2 + - y Mg 2 + libre </td><td></td><td></td><td></td></tr><tr><td> Solución salina equilibrada de Hank (HBSS) con rojo fenol </td><td> Fisher Scientific </td><td> SH3001603 </td><td></td></tr><tr><td> De bicarbonato de sodio </td><td> Sigma </td><td> S6014 </td><td></td></tr><tr><td> HEPES 1M en NaCl al 0,85% </td><td> Lonza </td><td> 17-737E </td><td></td></tr><tr><td> Suero fetal bovino (SFB) </td><td> Atlanta biológicos </td><td> S11150H </td><td> Inactivado por calor </td></tr><tr><td> EDTA 0,5 M (pH 8,0) </td><td> Cellgro </td><td> 46 a 034-CI </td><td></td></tr><tr><td> Collagenase tipo VIII </td><td> Sigma </td><td> C2139 </td><td></td></tr><tr><td> DNasa I </td><td> Roche </td><td> 14785000 </td><td> Una solución de archivo: 100mg/ml </td></tr><tr><td> LIVE / DEAD Se fija de Aqua kit de Dead Cell Mancha para la excitación de 405 nm </td><td> Invitrogen </td><td> L34957 </td><td> Utilice a 1:1000 </td></tr><tr><td> CD45-PerCP MAB (30F11) </td><td> BD </td><td> 557235 </td><td> Utilice a 1:100 </td></tr><tr><td> CD103-PE MAB (M290) </td><td> BD </td><td> 557495 </td><td> Utilice a 1:100 </td></tr><tr><td> FcγRIII / II MAB (2.4G2) </td><td> BD </td><td> 553141 </td><td> El uso de 1:200 </td></tr><tr><td> CD11c-APC MAB (N418) </td><td> eBioscience </td><td> 17-0114-82 </td><td> Utilice a 1:100 </td></tr><tr><td> MHC-II (I-Ab)-Alexa Fluor 700 MAB </td><td> eBioscience </td><td> 56-5321-82 </td><td> Utilice a 1:100 </td></tr><tr><td> CD11b-450 eFluorMAB (M1/70) </td><td> eBioscience </td><td> 48-0112-82 </td><td> El uso de 1:200 </td></tr><tr><td> F4/80-PE-Cy7 MAB (BM8) </td><td> eBioscience </td><td> 25-4801-82 </td><td></td></tr><tr><td> CD11b microperlas </td><td> Miltenyi Biotec </td><td> 130-049-601 </td><td></td></tr><tr><td> CD11c microperlas </td><td> Miltenyi Biotec </td><td> 130-052-001 </td><td></td></tr><tr><td> 50 ml tubos cónicos </td><td> BD Falcon </td><td> 352098 </td><td></td></tr><tr><td> Sola malla colador de alambre </td><td> Chefmate </td><td></td><td></td></tr><tr><td> Pequeño barco de peso </td><td> Fisher Scientific </td><td> 08-732-116 </td><td></td></tr><tr><td> 100 micras filtro de células </td><td> BD Falcon </td><td> 352360 </td><td></td></tr><tr><td> 40 micras filtro de células </td><td> BD Falcon </td><td> 352340 </td><td></td></tr><tr><td> 5 mL de poliestireno de fondo redondo tubos </td><td> BD Falcon </td><td> 352235 </td><td> Utilice a 1:100 </td></tr><tr><td> MaxQ 4450 de sobremesa agitador orbital </td><td> Thermo Scientific </td><td></td><td></td></tr><tr><td> LS MACS columna </td><td> Miltenyi Biotec </td><td> 130-042-401 </td><td></td></tr><tr><td> LSR II </td><td> BD </td><td></td><td></td></tr><tr><td> FACSAria II </td><td> BD </td><td></td><td></td></tr></tbody></table>

isolation and characterization of dendritic cells and macrophages from the mouse intestine

Dentro del intestino residen poblaciones únicas de las células inmunitarias innatas y adaptativas que están involucrados en la promoción de la tolerancia hacia la flora comensal y antígenos alimentarios, mientras que concomitantemente a punto restante para montar las respuestas inflamatorias hacia el 1,2 patógenos invasores. Células presentadoras de antígeno, en particular los PED y los macrófagos, juegan un papel crítico en el mantenimiento de la homeostasis inmune intestinal a través de su capacidad de detectar y responder apropiadamente a la microbiota 3-14. El aislamiento eficiente de los países en desarrollo y los macrófagos intestinales es un paso fundamental en la caracterización del fenotipo y la función de estas células. Mientras que muchos métodos eficaces de aislar intestinales células inmunes, incluyendo los DC y macrófagos, han sido descritos 6,10,15-24, muchas veces se basan en digestiones largas que pueden influir negativamente superficie celular expresión del antígeno, la viabilidad celular, y / o el rendimiento de la célula. Aquí, detalles de una metodología para el aislamiento rápido de un gran número de viable, los países en desarrollo y los macrófagos intestinales. Caracterización fenotípica de los DC intestinales y macrófagos se lleva a cabo por tinción aislados directamente con las células intestinales específicas fluorescencia-etiquetados anticuerpos monoclonales para el análisis de flujo de múltiples colores citometría. Por otra parte, CC de alta pureza y de las poblaciones de macrófagos están aislados de los estudios funcionales utilizando CD11c y CD11b magnética activadas por cuentas de células de clasificación seguido por la selección de células.

Watch this Scientific Journal Video about Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine at JoVE.com

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Aquí, le detallamos una metodología para el aislamiento rápido de las células dendríticas de ratones intestinales (DCS) y los macrófagos. Caracterización fenotípica de los países en desarrollo y los macrófagos intestinales se realiza utilizando varios colores, mientras que el flujo de análisis de citometría de enriquecimiento de bolas magnéticas seguido por la selección de células se utiliza para producir poblaciones de alta pureza para los estudios funcionales.

Aislamiento y caracterización de las células dendríticas y los macrófagos del intestino delgado de ratón

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

isolation-characterization-dendritic-cells-macrophages-from-mouse

Research

JoVE Journal

Immunology and Infection

38.9K vistas.  Emory University. Aquí, le detallamos una metodología para el aislamiento rápido de las células dendríticas de ratones intestinales (DCS) y los macrófagos. Caracterización fenotípica de los países en desarrollo y los macrófagos intestinales se realiza utilizando varios colores, mientras que el flujo de análisis de citometría de enriquecimiento de bolas magnéticas seguido por la selección de células se utiliza para producir poblaciones de alta pureza para los estudios funcion...

Video: Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Aislamiento de células dendríticas del pulmón de ratón

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Investigación

Inmunología e Infección

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

Aislamiento de células dendríticas mieloides y células epiteliales de timo humano

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

Células T Captura bacterias por transinfection de células dendríticas

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Aislamiento y análisis de citometría de flujo de células inmunes del cerebro isquémico Ratón

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Generación e identificación de GM-CSF Derivado alveolares-como los macrófagos y las células dendríticas A partir de médula ósea de ratones

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Caracterización de las células dendríticas derivadas de monocitos humanos mediante imágenes de Citometría de Flujo: una comparación entre dos protocolos de aislamiento de monocitos

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

Caracterización fenotípica de los macrófagos a partir de riñón de rata por Citometría de Flujo

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Clasificación de células activadas por fluorescencia para la purificación de células dendríticas plasmocitoide de la médula ósea de ratones

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Generación de células dendríticas derivadas de monocitos humanos de sangre entera

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.