Este trabajo fue financiado por el piloto y la subvención Estudio de Viabilidad de la UCLA-CURE Center, la enfermedad de Crohn y Colitis Foundation of America Award de Desarrollo Profesional (# 2691) y el Instituto Nacional de Salud NIDDK K01 (DK084256) la financiación a Hon Wai Koon. La médula ósea operación irradiación fue asistido por Bernard Levin y Cocina Scott de UCLA Center for AIDS Research Mouse / instalaciones Humanos Core Chimera. Operación de citometría de flujo fue asistido por instalaciones UCLA Core Vector.

1. Antes-de-empezar Consideraciones técnicas <ol><li> Le recomendamos que use C57/BL6 ratones de tipo salvaje y ratones knockout para el experimento porque los ratones con moléculas de CD correspondientes pueden adquirirse de los proveedores más importantes del ratón. </li><li> Puesto que es difícil realizar un seguimiento de los ratones de tipo salvaje y de ratones knockout derivados células del donante después del trasplante de médula ósea, es necesario utilizar CD45.1 ratones para representar ratones de tipo salvaje. (CD45.1 C57/SJL PESO ratones Jackson Laboratorios de stock # 002014). </li><li> La mayoría de las colonias de ratones son CD45.2. Nuestros ratones knockout pertenecen a CD45.2 genotipo (o bien una fuente CD45.2 ratones WT: Jackson Laboratories stock # 000664). La determinación de CD45.1 o CD45.2 genotipo se puede realizar mediante citometría de flujo de la médula ósea y la sangre periférica como se ha mencionado en la sección 7 de este protocolo. </li><li> Los ratones después de la irradiación han puesto en peligro la inmunidad. Mantener los ratones en instalación libre de patógenos. </li><li> Funcionamiento del irradiador requiere especial clearanc seguridade y formación. Para ahorrar tiempo en los procesos de formación y aprobación, lo mejor es colaborar con un laboratorio con personal cualificado que sea capaz de manejar el irradiador. </li><li> Muchas instituciones cuentan con laboratorios de citometría de flujo con técnicos experimentados. Para minimizar los problemas de funcionamiento de citometría de flujo, es mejor discutir su plan de citometría de flujo experimentar con el técnico con experiencia antes de empezar. </li><li> Los ratones fueron asignados de esta manera (10 ratones por grupo): </li></ol><table border="1"><tbody><tr><td> Grupo </td><td></td><td> Donante de médula ósea para el destinatario </td><td> Tratamiento </td></tr><tr><td> La </td><td> BM intercambio </td><td> CD45.1 WT KO CD45.2 </td><td> DSS colitis </td></tr><tr><td> B </td><td></td><td></td><td> Agua control normal </td></tr><tr><td> C </td><td> BM intercambio </td><td> CD45.2 KO a CD45.1 PESO </td><td> DSS colitis </td></tr><tr><td> D </td><td></td><td></td><td> Agua control normal </td></tr><tr><td> E </td><td> Falso </td><td> CD45.1 WT para CD45.1 PESO </td><td> DSS colitis </td></tr><tr><td> F </td><td></td><td></td><td> Agua control normal </td></tr><tr><td> G </td><td> Falso </td><td> CD45.2 KO KO a CD45.2 </td><td> DSS colitis </td></tr><tr><td> H </td><td></td><td></td><td> Agua control normal </td></tr></tbody></table> * Una breve ilustración del protocolo experimental se muestra en la figura 1. 2. Los ratones receptores irradiación <ol><li> Los ratones son criados y mantenidos en instalaciones libres de patógenos. Coloque los ratones en pie irradiación autoclave con filtro. Coloque un ratón en cada ranura de pastel de irradiación. Coloque el pastel en el irradiador y asegúrese de que el plato giratorio y el pastel está dando vuelta. </li><li> Encienda la bomba de airepara la ventilación. Cierre la puerta del irradiador y bloquearlo. </li><li> Irradiar a los ratones con 1.000 rad (que es equivalente a 10 Gy) alrededor de 10 min (dependiendo de la fuente de radiación y la vida media-). Desde este punto de tiempo, los ratones irradiados son inmunodeficientes y tienen inmunidad débil contra la infección. Después de la irradiación, tomar la tarta y colocar en un recipiente estéril para transportarlo al gabinete de bioseguridad en las instalaciones de los animales. Evite la exposición de los ratones con el medio ambiente externo para minimizar las posibilidades de infección. </li><li> En gabinete de bioseguridad, la transferencia a las jaulas de los ratones del ratón autoclave autoclave con ropa de cama (4 ratones por jaula Agregue agua estéril nueva suspensión Sulfatrim -. 3,12 ml por 100 ml de agua). </li><li> Envuelva la botella de agua con papel de aluminio, como los antibióticos son sensibles a la luz. Agitar para mezclar la botella de solución Sulfatrim bien antes de cargarlo a la jaula. </li></ol> 3. Donantes de Médula Ósea de extracción <ol><li> Sacrificar los ratones con dióxido de carbono o isoflurmiento y luego sumergir en solución 1:200 Lysol. Rocíe los ratones con 70% de etanol para humedecer la piel y diseccionar huesos abiertos. </li><li> Remojar los instrumentos de disección en 70% de etanol para la esterilización. </li><li> Realizar la disección en un gabinete de bioseguridad. Utilizar los instrumentos de disección estéril para exponer el húmero, el fémur, la tibia y el peroné huesos, libre de fijación de los músculos y ligamentos. </li><li> Cortar abrir ambos extremos de los huesos para mostrar la médula ósea roja. Mantenga los huesos con fórceps. Lavar la médula ósea roja con PBS frío con jeringa de 3 ml y agujas de 25G a una placa de Petri. Enjuague de ambos extremos de los huesos para producir células de la médula ósea más. </li><li> Use 6 ml jeringas y agujas 18G1 / 2 a romper los tapones rojos de médula ósea por aspiración repetida y expulsión. Transferencia de médula ósea con 50 ml tubos Falcon. </li><li> Girar la médula ósea en 1.800 rpm durante 5 min a 4 ° C. Desechar el sobrenadante, resuspender el sedimento por agitación durante 5-10 segundos. </li><li> Diluir el tampón de lisis de RBC 10 veces con agua estéril. Anunciod 1X tampón de lisis RBC (5 ml por ratón donante) y agitar de nuevo durante 5 segundos y mantener exactamente 3 minutos a temperatura ambiente. </li><li> Después de 3 min, llenar los tubos con 20 ml de PBS frío para detener la lisis y mezclar. Verter el contenido a través de un filtro de células 40 micras directamente en un nuevo tubo Falcon de 50 ml. </li><li> Enjuague los tubos originales con 5 ml de PBS y transferir a filtro de células así. Completar hasta 50 ml con PBS y mezclar. </li></ol> 4. Contando Bone Marrow Donor células <ol><li> Añadir 100 l de azul de tripano a un tubo Eppendorf y 100 l de suspensión de médula ósea a un tubo Eppendorf y se mezcla. Pipetear la mezcla de células teñidas con un hemocitómetro. </li><li> La cantidad de células se calcula por recuento total de células de médula ósea de estar en los tubos Falcon = número de células sin teñir en 16 cuadrados x 2 (debido a la dilución Trypan azul) x 10.000 x 50 ml </li><li> Decantar las células de médula ósea en 50 ml en tubos Falcon de 2.000 rpm durante 5 min a 4 ° C. </li><li> Eliminar el sobrenadante. Basadoen el total de células, se resuspende el pellet con PBS a 1 x 10 8 células por ml </li></ol> 5. Infusión a ratones receptores <ol><li> Calentar los ratones receptores sobre una almohadilla de calor y bajo una lámpara de calentamiento. Ponga los ratones receptores de inmovilización. Inyectar 1 x 10 7 células por ratón en 100-200 l por vía intravenosa. </li><li> La inyección de médula ósea de un donante se debe hacer entre 4-24 horas después de la irradiación. </li><li> Manténgase 4 ratones inyectados por jaula. Mantener los ratones receptores inyectados en jaulas autoclave con agua tratada con antibióticos durante las primeras 4 semanas en las instalaciones patógeno animal libre y dejar que los ratones recuperar la inmunidad. Cambio jaulas, tratado con antibióticos en aguas y alimentos cada 4 días para mantener la higiene. </li></ol> 6. La inducción de la colitis y la evaluación de la colitis <ol><li> 4 semanas después del trasplante de médula ósea y la irradiación, cambiar a agua normal sin antibióticos y mantener los ratones durante 2 semanas más para recuperar su normalidadla microflora intestinal. </li><li> 6 semanas después de la inyección de médula ósea, medir el peso corporal inicial. Algunos ratones se les da 5% de sulfato de dextrano en beber agua ad libitum para inducir la colitis. Algunos ratones se les da agua potable regular sólo como grupos de control normales. </li><li> 5 días después, extraer sangre periférica, traslado a tubos recubiertos de heparina (Vacutainer) y mantener en hielo. Piscina de la sangre de los ratones 4 a 1 metro. Combine 300 l de sangre con 300 l de buffer de tinción celular = 600 l. Divida la sangre a 5 tubos Eppendorf, cada uno con 100 l. </li><li> Sacrificar los ratones con dióxido de carbono o isoflurano. Evaluar resultados macroscópicos daños (por favor refiérase a otra publicación vídeo JOVE para obtener más información 1). Medir el cambio de peso corporal. Medir la longitud del intestino grueso y con calibrador digital. Observe textura heces (dura, blanda o con sangre). Determinar sangre oculta en las heces con Hemooccult. Disecciona tejidos de colon y fijar en formol para H &amp; E tinción. </li><li> Disecciona un fémur bone por ratón, se extrae médula ósea y enjuagar la médula ósea con PBS frío a través de aguja 25G y una jeringa de 1 ml. De 4 estrella médula ósea se conecta a un tubo y mantener en hielo. </li><li> Verter la médula ósea a una placa de Petri, la cizalladura del tapón de médula ósea con aguja 18G y 5 ml jeringa varias veces hasta que no enchufe médula ósea está presente. </li><li> Decantar con 1.500 rpm durante 5 min a 4 ° C. Remover el sobrenadante y resuspender con 600 l tampón de tinción celular. Divida la médula ósea se resuspendieron a 5 tubos Eppendorf, cada uno con 100 l. </li></ol> 7. Inspección de Calidad de Trasplante de Médula Ósea por citometría de flujo <ol><li> Etiquete cada tubo de muestra de sangre o de médula ósea # 1-5: </li><li> Preparar la mezcla de anticuerpos para cada tubo respectivo en la oscuridad. </li></ol> # 1 No anticuerpo # 2 30 l control de isotipo FITC + 30 mu l PE control de isotipo + 15 mu l CD16/32 bloqueo # 3 30 l FITC CD45.1 Ab + 15 mu l CD16/32 bloqueo # 4 30 l PECD45.2 Ab + 15 mu l CD16/32 bloqueo # 5 30 l FITC CD45.1 Ab + 30 l PE CD45.2 Ab + 15 mu l CD16/32 bloqueo <table border="1"><tbody><tr><td> Añadir a nada muestra de sangre o de médula ósea # 1 </td></tr><tr><td> Añadir 5 l de mezcla de anticuerpos # 2 para cada muestra de sangre o de médula ósea # 2 </td></tr><tr><td> Añadir 3 l de mezcla de anticuerpos # 3 para cada muestra de sangre o de médula ósea # 3 </td></tr><tr><td> Añadir 3 l de mezcla de anticuerpos # 4 para cada muestra de sangre o de médula ósea # 4 </td></tr><tr><td> Añadir 5 l de mezcla de anticuerpo n º 5 para cada muestra de sangre o de médula ósea # 5 </td></tr></tbody></table><ol start="3"><li> Mantener en la oscuridad en hielo durante 30 min. </li><li> Añadir 2 ml de tampón de lisis 1X RBC a cada tubo. Mantener en la oscuridad en hielo durante 15 min. </li><li> Decantar las células a 1.500 rpm, 5 min a 4 ° C. (GH 3,8 rotor, 1.500 rpm = 350 xg) Remover el sobrenadante. Resuspender el precipitado con 500 l tampón de tinción de las células en la oscuridad brevemente, a continuación, vórtice. </li&gt; <li> Ver el CD45.1 (representando WT) y CD45.2 (representando KO) en la sangre inmunoteñida y muestras de médula ósea por citometría de flujo. Seleccione FITC para representar PESO CD45.1 y PE para representar KO CD45.2. </li><li> Analizar los resultados de la citometría de flujo por software FlowJo. Calcular la relación de FITC: PE o PE relación: relación de FITC para determinar si el genotipo donante es el genotipo predominante en la sangre y la médula ósea. </li></ol>

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No hay conflictos de interés declarado.

Este enfoque trasplante de médula ósea es adecuado para la investigación de la inmunología colitis, infección, cáncer, obesidad y otras enfermedades. Este experimento de trasplante de médula ósea es necesaria cuando el gen de interés se expresa en tanto derivadas de médula ósea y no células de la médula ósea derivados y el gen de interés se sospecha para mediar la enfermedad por células de cualquiera de la población. Por ejemplo, péptido antimicrobiano de catelicidina está demostrado que modulan la co...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Célula tampón de tinción </td><td> BioLegend </td><td> # 420201 </td></tr><tr><td> 10X tampón de lisis RBC </td><td> BioLegend </td><td> # 420301 </td></tr><tr><td> FITC ratón isotipo de control </td><td> BioLegend </td><td> # 400207 </td></tr><tr><td> PE ratón isotipo de control </td><td> BioLegend </td><td> # 400211 </td></tr><tr><td> PE anti-ratón CD45.2 </td><td> BioLegend </td><td> # 109807 </td></tr><tr><td> FITC anti-ratón de CD45.1 </td><td> BioLegend </td><td> # 110705 </td></tr><tr><td> Anti-ratón CD16/32 bloqueo </td><td> BioLegend </td><td> # 101301 </td></tr><tr><td> 40 filtro de células micras </td><td> Fisherbrand </td><td> # 22363547 </td></tr><tr><td> PBS 1X </td><td&gt; MP Biomedicals </td><td> # 1860454 </td></tr><tr><td> La heparina </td><td> Fisherbrand </td><td> # BP2425 </td></tr><tr><td> Sulfatrim (SMZ) </td><td> Qualitest </td><td> NC9242720 (pescador) </td></tr><tr><td> Lysol IC </td><td> Andwin Científico </td><td> NC9745686 (pescador) </td></tr><tr><td> Vaccutainer </td><td> BD </td><td> # 8000813 </td></tr><tr><td> Ratón restrainer </td><td> Braintree Scientific </td><td> # TV-150 </td></tr><tr><td> Irradiador </td><td> JL Pastor y Asociados </td><td> Mark I 68A </td></tr><tr><td> Citómetro de flujo </td><td> BD </td><td> BD FACSCanto II </td></tr><tr><td> Citómetro de flujo de tubo de ensayo </td><td> Halcón </td><td> # 352052 </td></tr><tr><td> Calibre digital </td><td> Fisherbrand </td><td> 14-648-17 </td></tr><tr><td> Hemoccult TIC </td><td> Beckman Coulter </td><td> G0328QW </td></tr&gt; </tbody></table>

differentiating functional roles of gene expression from immune and non-immune cells in mouse colitis by bone marrow transplantation

Para entender el papel de un gen en el desarrollo de colitis, se compararon las respuestas de los ratones de tipo salvaje y el gen de interés ratones knockout deficientes a colitis. Si el gen de interés se expresa tanto en las células derivadas de la médula ósea y no células de médula ósea derivadas del huésped, sin embargo, es posible diferenciar el papel de un gen de interés en células de médula ósea derivadas de médula y no óseo células derivadas mediante la técnica de trasplante de médula ósea. Para cambiar la médula ósea procedentes de células genotipo de los ratones, la médula ósea original de los ratones receptores fueron destruidas por irradiación y luego se sustituye por la nueva médula ósea del donante de genotipo diferente. Cuando de tipo salvaje ratones donantes de médula ósea se trasplantan a ratones knockout, se podría generar ratones knockout con la expresión del gen de tipo salvaje en células de médula ósea derivadas. Por otra parte, cuando los donantes ratones knockout médula ósea trasplantadas a ratones receptores de tipo salvaje, de tipo salvaje ratones sin el gen de interés expresarde la médula ósea células derivadas fueron producidos. Sin embargo, el trasplante de médula ósea puede no ser 100% completo. Por lo tanto, hemos utilizado cúmulo de diferenciación (CD) moléculas (CD45.1 y CD45.2) como marcadores de las células del donante y del receptor para realizar un seguimiento de la proporción de células de la médula ósea de un donante derivados en los ratones receptores y el éxito de los trasplantes de médula ósea. De tipo salvaje ratones con genotipo CD45.1 ratones knockout y con CD45.2 genotipo fueron utilizados. Después de la irradiación de ratones receptores, las células donantes de médula ósea de diferentes genotipos fueron infundidas en los ratones receptores. Cuando la nueva médula ósea regenerada para hacerse cargo de su inmunidad, los ratones se expusieron por agentes químicos (sulfato de dextrano sódico, DSS al 5%) para inducir la colitis. Aquí también mostró el método para inducir la colitis en ratones y evaluar la función del gen de interés expresado a partir de células de médula ósea derivadas. Si el gen de interés a partir de las células óseas derivadas juega un papel importante en el desarrollo de la enfermedad (tal como Escherichiatis), el fenotipo de los ratones receptores con trasplante de médula ósea puede ser alterado de manera significativa. Al final de los experimentos de colitis, la médula ósea derivada células en la sangre y la médula ósea se marcaron con anticuerpos contra CD45.1 y CD45.2 y su proporción cuantitativa de la existencia podría ser usado para evaluar el éxito del transplante de médula ósea por citometría de flujo. Éxito del trasplante de médula ósea debe mostrar una gran mayoría de genotipo donante (en término de marcador molécula de CD) a lo largo genotipo receptor tanto en la médula ósea y la sangre de los ratones receptores.

Si el gen de interés desempeña un papel significativo en las células inmunes durante el desarrollo de la colitis, los ratones que recibieron médula ósea de genotipo diferente a través de trasplante de médula ósea (WT a KO o KO a WT) debería tener una respuesta alterada a la colitis DSS. Uno de los parámetros más importantes para la determinación de la gravedad de la colitis es la tinción H &amp; E de los tejidos de colon. Colon cambios en la estructura de tejido y los signos de la inflamación pueden evalua...

Watch this Scientific Journal Video about Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation at JoVE.com

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

Trasplante de médula ósea proporciona una manera de cambiar el genotipo de las células de médula ósea derivadas. Si el gen de interés se expresa tanto en las células derivadas de la médula ósea y no células de médula ósea derivadas, transplante de médula ósea puede cambiar las células de médula ósea derivadas de un genotipo diferente sin cambiar la no de la médula ósea derivada genotipo celular.

Diferenciar los roles funcionales de la expresión génica de las células inmunes y no inmunes en la colitis de ratón por Trasplante de Médula Ósea

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

differentiating-functional-roles-gene-expression-from-immune-non

Research

JoVE Journal

Immunology and Infection

17.3K vistas.  The University of California Los Angeles, Los Angeles. Trasplante de médula ósea proporciona una manera de cambiar el genotipo de las células de médula ósea derivadas. Si el gen de interés se expresa tanto en las células derivadas de la médula ósea y no células de médula ósea derivadas, transplante de médula ósea puede cambiar las células de médula ósea derivadas de un genotipo diferente sin cambiar la no de la médula ósea derivada genotipo celular.

Video: Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

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 ...

Investigación

Inmunología e Infección

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

Aislamiento, Purificación y Etiquetado de neutrófilos de médula ósea de ratón de Estudios funcionales y experimentos de transferencia adoptivos

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Seguimiento de médula ósea de ratón monocitos<em&gt; En Vivo</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

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. ...

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 ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Identificación y aislamiento de Oligopotent y linaje comprometido progenitores mieloides de la médula ósea de ratón

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

Aislamiento de células mononucleares lamina Propria de Colon Murino usando colagenasa E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient&#39;s survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

Plataforma de trasplante de médula ósea para investigar el papel de las células dendríticas en la enfermedad de injerto contra huésped

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

Procedimientos de trasplante de médula ósea en ratones para estudiar la hematopoyesis clonal

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to proteins of interest. Identification of gene functions in cell line models is important for gene discovery and exploration of cell-intrinsic mechanisms. However, genetic manipulations of immune cells such as T cells and macrophage cell lines using CRISPR/Cas9-mediated knock-in are difficult because of the low transfection efficiency of these cells, especially in a quiescent state. To modify genes in immune cells, drug-resistance selection and viral vectors are typically used to enrich for cells expressing the CRIPSR/Cas9 system, which inevitably results in undesirable intervention of the cells. In a previous study, we designed dual fluorescent reporters coupled to CRISPR/Cas9 that were transiently expressed after electroporation. This technical solution leads to rapid gene deletion in immune cells; however, gene knock-in in immune cells such as T cells and macrophages without the use of drug-resistance selection or viral vectors is even more challenging. In this article, we show that by using cell sorting to aid selection of cells transiently expressing CRISPR/Cas9 constructs targeting the Rosa26 locus in combination with a donor plasmid, gene knock-in can be achieved in both T cells and macrophages without drug-resistance enrichment. As an example, we show how to express human ACE2, a receptor of SARS-Cov-2, which is responsible for the current Covid-19 pandemic, in RAW264.7 macrophages by performing knock-in experiments. Such gene knock-in cells can be widely used for mechanistic studies.

This protocol uses fluorescent reporters and cell sorting to simplify knock-in experiments in macrophage and T cell lines. Two plasmids are used for these simplified knock-in experiments, namely a CRISPR/Cas9- and DsRed2-expressing plasmid and a homologous recombination donor plasmid expressing EBFP2, which is permanently integrated at the Rosa26 locus in immune cells.

Knock-in de genes por CRISPR/Cas9 y clasificación celular en líneas de macrófagos y células T

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to ...