This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC, IG14593, MFAG13235), the Fondazione Cariplo (Grant 2010-0678 and NANOVAC) and the Fondazione Regionale per la Ricerca Biomedica.

de aprobación del estudio: Los protocolos experimentales fueron aprobados por el Ministerio de Sanidad italiano (Roma, Italia) de acuerdo con el Decreto Legislativo 27 de Enero de 1992, n. 116 &quot;attuazione della Direttiva n. 86/609 / CEE en materia di protezione degli animali utilizzati un fini sperimentali o ad altri Fini scientifici.&quot; 1. Obtención de las muestras de piel <ol><li> La eutanasia el ratón por dislocación cervical. </li><li> Oreja: <ol><li> Cortar la parte sin pelo de las orejas del ratón. </li><li> dorsal separada y parte ventral mediante tracción con fórceps. Eliminar cualquier resto de cartílagos, raspando suavemente la parte interna de las orejas con fórceps. </li></ol></li><li> la piel del tronco: <ol><li> Afeitarse toda la piel del animal, tanto con una máquina de afeitar eléctrica y con la loción depilatoria delicada. </li><li> Cortar la muestra de piel deseado (de 2 cm 2 hasta toda la piel de los animales). <ol><li> Si cortando todo el dorsal y nclusotral de la piel del ratón, haga un corte vertical en el medio del ratón de nuevo y luego se corta a lo largo de las fronteras de las áreas afeitadas de todo el cuerpo del ratón. </li><li> separar suavemente la piel del peritoneo y músculos de la espalda, manteniendo la muestra de piel todavía con el fórceps, mientras que la separación de la muestra de piel con la punta de bordes redondeados cerrado de un tijeras quirúrgicas o un bisturí. </li></ol></li><li> Preparar una placa de cultivo de 100 mm que contiene 10 ml de PBS frío. </li><li> Eliminar la grasa subcutánea sumergiendo la muestra de piel en PBS en la placa preparada en el paso 1.3.3 y raspando la muestra de piel con pinzas. </li></ol></li><li> Cola: <ol><li> Cortar la cola del ratón. </li><li> Hacer un corte vertical en la cola con unas tijeras quirúrgicas o bisturí quirúrgico, a partir de la base de la cola y que llegan hasta la punta de la cola. </li><li> El uso de fórceps para separar la piel de la cola. Agarrar el borde del corte en la base de la cola y tire suavemente hacia la punta, mientras que la celebración de lacuerpo de la cola con unas pinzas. </li><li> Eliminar cualquier exceso de grasa, raspando suavemente la muestra de piel. </li></ol></li><li> Alfombra de pies <ol><li> Cortar todo el pie ratón con tijeras quirúrgicas. Evitar cortar cualquier piel con pelo. </li><li> Cortar la piel del pie longitudinalmente desde el tobillo hasta el comienzo de los dígitos. </li><li> Mantenga los huesos del tobillo con unas pinzas o con la mano, mientras que agarrar la piel con pinzas y tire de la piel hacia los dígitos como si quitándose un guante. </li></ol></li></ol> 2. Digestión <ol><li> Preparar el cóctel de la digestión de la solución madre (preparado como en la Lista de Materiales) con la siguiente receta: Mezcla de Enzimas (ver Lista de Materiales para las especificaciones) 300 g / ml, Dnase1 50 U / ml. Diluir en RPMI 5% de FBS. </li><li> Picar las muestras de piel con tijeras en trozos pequeños en una placa de Petri de 35 mm con 2 ml de digestión de cóctel (Para la piel del tronco: 2 ml de cóctel Digestión cada 2 cm2 de piel, hasta 3 ml en total). </li><li>Se incuba a 37 ° C durante 90 minutos. Agitar suavemente la placa de Petri 1-2 veces durante la incubación. </li><li> Verter muestras de piel digeridos en un colador de 70 micras de células en 66 mm plato de Petri que contiene 1 ml de RPMI1640 5% de FBS. </li><li> Lavar el filtro con 5 ml de RPMI 1640 5% de FBS. </li><li> Exprimir la muestra a través del filtro de células con un émbolo de la jeringa. </li><li> émbolo de color, placa de Petri y el filtro con 20 ml de RPMI1640 FBS al 5%, la transferencia en un tubo de 50 ml. </li></ol> 3. Los anticuerpos y Etiquetado <ol><li> Se lava la suspensión obtenida dos veces con 5 ml de PBS 1% de SFB (o BSA). </li><li> Etiqueta de la suspensión celular obtenida con los anticuerpos deseados. <ol><li> Resuspender las muestras en 100 l / muestra de una mezcla que contiene 2 mg / ml de aCD16 / CD32 (clon 2.4G2) diluido en PBS 1% de FBS. </li><li> Incubar durante 15 min a 4 ° C con el fin de bloquear la unión no específica de los anticuerpos deseados a través de receptores de Fc. </li><li> Añadir los anticuerpos indicados en la tabla1. <ol><li> Preparar una mezcla de diluir los anticuerpos deseados en 100 l / muestra de PBS 1% de FBS a una concentración de 4 mg / ml. </li><li> Añadir 100 ml de la mezcla preparada en la etapa 3.2.3.1 a cada muestra de manera que en el tubo, habrá un volumen de 200 l de mezcla de anticuerpos a una concentración final de 2 mg / ml. </li></ol></li><li> Incubar durante 30 minutos a 4 ° C y la cubierta de la luz. </li></ol></li><li> Lavar con 1 ml de PBS 1% de FBS, de centrifugación durante 5 min a 400 xg, descartar el sobrenadante y resuspender en 300 l de PBS 1% de FBS. </li><li> Añadir DAPI a una concentración final de 2 mg / ml y se incuba a 4 ° C, protegido de la luz durante 10 minutos. </li><li> Analizar las muestras obtenidas por FACS. <ol><li> Con el fin de aislar los leucocitos, utilizar la siguiente estrategia gating: <ol><li> En primer lugar eliminar las células muertas, colocando puertas en la población DAPI-. </li><li> Seleccione singletes en el FSC-A vs. parcela FSC W como se indica en la Figura 1 </strong&gt;. </li><li> Puerta de las células en función del tamaño y la granularidad típico de los leucocitos de interés. </li><li> Seleccionar CD45 + células en el FSC-A parcela CD45 como se indica en la Figura 1. </li></ol></li><li> Distinguir las diferentes poblaciones de leucocitos por la expresión de marcadores de superficie como sigue: <ol><li> Identificar los países en desarrollo como células CD11c + II + MHC. Estos pueden ser analizados después para la expresión de CD207 para distinguir las células de Langerhans y CD207 + dérmica DCs de CD207- dérmica DCs. </li><li> Identificar los macrófagos como células CD64 + F4 / 80 +. </li><li> Identificar las células B como las células CD19 + MHCII +. </li><li> Identificar las células T como CD8 + o células CD4 +. </li></ol></li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Hemos descrito un método para la preparación de suspensiones de una sola célula a partir de diferentes regiones de la piel del ratón. El método de la digestión hemos adoptado, no sólo da altos rendimientos sino que también conserva la expresión de marcadores de superficie, que es fundamental para el posterior análisis de FACS. El uso de un cóctel de colagenasa I y II y termolisina, garantiza lote mínimo a las diferencias de lote en la actividad enzimática haciendo de este método altamente reproducible. Otros métodos publicados se basan en diferentes cócteles de enzimas, pero en nuestras manos que dan rendimientos más bajos y, más importante aún, son menos reproducibles. Nuestro método, sin embargo, no discrimina entre las células que pueblan la epidermis y las células que pueblan la dermis. Dado que en muchos casos podría ser útil distinguir entre estos dos compartimentos diferentes, nos dirigimos al lector a estos trabajos previos que utilizan tales métodos. 15 En nuestra muestra la experiencia de la piels de la oreja y la cola son más fáciles de digerir que la piel del tronco, donde la capa de grasa subcutánea tiene que ser raspado a fondo a fin de que la dermis accesible para el cóctel de enzimas. El paso-de engrase, sin embargo, así como el corte debe realizarse de forma rápida y suavemente a fin de mantener la viabilidad celular. Una etapa de desengrasado prolongada o demasiado a fondo puede provocar daños en el residente células inmunes en la piel y por lo tanto resultar en un rendimiento mucho más bajo y la disminución de la viabilidad de las células recuperadas. También hay que señalar que para preservar la integridad de los marcadores de superficie, la digestión se debe realizar en un medio carente de agentes tales como β-mercaptoetanol reductor. Para evitar daños no deseado a los marcadores de superficie, el tiempo de incubación debe mantenerse lo más corto posible y, en cualquier caso, no prolonga durante más de 90 min. Debe prestarse especial atención, también, tomada al verter las muestras de piel digeridos en la célulacolador. Es importante lavar a fondo la piel digerido con el fin de lavar las células liberadas antes de romper la muestra digerida con el émbolo de la jeringa. Con este paso, la muestra de piel se ha limpiado de las células inmunes liberados luego de que no se someten a ningún tipo de estrés más físico. El aplastamiento se debe también realizar con cuidado a fin de no destruir cualquier célula y tanto el filtro de células y el émbolo de la jeringa se deben lavar a fondo en la etapa de lavado posterior. la digestión de la piel también se puede realizar en un Falcon de 50 ml en un baño de agua a 37 ° C. En este caso la piel puede ser cortado de antemano y se coloca en el tubo Falcon. Tenga en cuenta que las muestras de piel siempre deben ser sumergidos en tampón de digestión durante la incubación y que oído-piezas se adhieren a los lados de tubos Falcon. Al utilizar tubos Falcon, por lo tanto, asegúrese de que las piezas de piel picada estancia sumergidos en el medio de la digestión. Este método puede ser útil en many diferentes parámetros experimentales, en particular cuando se estudia la piel no inflamado. Durante la inflamación, la vasodilatación y la hinchazón de los tejidos a aflojar la estructura del tejido y las células inmunes de la sangre son reclutados masivamente a la piel. La eficiencia de recuperación de células es, por lo tanto, mucho más alto. En condiciones homeostáticas, una manera eficaz para digerir la matriz de colágeno apretado de la dermis es, de hecho, necesario con el fin de recuperar la célula inmune que constituye la compleja red poblar la piel. El lector también podría estar interesado en una revisión exhaustiva que abarca el origen y el fenotipo de las diferentes células dendríticas y macrófagos pueblan la piel en ambas condiciones homeostáticas e inflamatorias. 16 En diferentes parámetros experimentales, diferentes porciones de piel de ratón se pueden tratar. Por ejemplo, en modelos de trasplante de piel, una porción de la cola podría ser trasplantado en el tronco de un animal que recibe; cuando studying formación de edema o procedimientos de inmunización, los estímulos son a menudo inyectaron en la almohadilla de la pata de los ratones, mientras que el oído se utiliza con frecuencia para evaluar la memoria de las células T con los ensayos de DTH. Con nuestro método podemos extraer eficazmente las células inmunes residentes de la piel de diferentes lugares haciendo nuestro método útil para el establecimiento experimental de elección.

The skin is one of the largest organs of the body and its large surface is continuously exposed to the external environment. Therefore the skin has to protect the organism from potential threats in order to maintain homeostasis, both by physical means, and by providing active protection toward potential pathogen entry. In a similar way to the gut and lung mucosa, the skin homes a variety of immune cells that continuously interact with the epithelium in order to maintain immune surveillance. This complex system that involves both immune and non-immune cells of the skin has been acknowledged since the early days of immunology, when in 1978 the term "Skin-associated lymphoid tissue" (SALT) 1 was first coined to describe such complexity. 
The large number of immune cells residing in the skin helps the organism not only to fight potential invasions, but also to orchestrate wound healing and to maintain tolerance toward self-antigens and the skin microbiota 2,3. 
Amongst skin resident immune cells, Dendritic Cells (DCs) have a crucial role in shaping immune responses and maintaining homeostasis 4. Dermal DCs and Langerhans cells readily respond to pathogen invasions and, upon migration to lymph nodes activate T cells and induce the expression of skin homing receptors on the newly generated effector T cells 5. DCs also have a fundamental role in regulating skin homeostasis. DCs migrating from the dermis to the lymph nodes in homeostatic conditions transport skin sequestered antigens with the purpose of inducing T cell tolerance mostly through the differentiation of regulatory T cells (Tregs) specific for skin antigens 6-8. 
T cells represent the most abundant population of immune cells in the skin. Not only do they infiltrate the skin during an infection but they also represent a stable population among skin resident lymphocytes 9,10. Both CD8+ and CD4+ resident memory (rm)T cells reside in the skin and, following an infection, can respond long before effector T cells are recruited from the blood. In the skin, also resides a population of tissue resident Tregs capable of maintaining tolerance toward skin antigens and tissue homeostasis. These cells are rapidly and potently activated after exposure to their cognate antigen and have therefore been defined as memory Tregs 11,12. 
Along with DCs and T cells, many innate immune cells, such as NK cells, gamma delta T cells, group 2 ILCs 13, Mast Cells and Macrophages, populate the skin and contribute to host protection. To analyze such a complex environment, it is mandatory to obtain single-cell suspensions from skin specimens with high efficiency while at the same time preserving the expression of surface markers for flow cytofluorimetric analysis or sorting. 
Murine skin presents an outer epidermal layer, constituted mainly of keratinocytes, Langerhans cells and dendritic epidermal T cells; and the dermal layer beneath. The dermis homes most of the immune cells and is made by an extracellular matrix in which collagen fibers are the most abundant, especially collagen-I and collagen-IV. Unlike human skin, mouse skin is covered in fur, and thus more populated with hair follicles. It is, also, much thinner than human skin and contains a muscular layer, the panniculus carnosus, which helps wound healing 14. These characteristics render it more difficult to efficiently disrupt the collagen net of the dermis in order to get immune cells out.
In this paper we provide a suitable method for digesting the extracellular matrix that relies on a cocktail of high purity collagenase 1 and 2 and thermolysin. Moreover, since the analysis of skin from different regions requires different experimental procedures, we will show different methods to efficiently obtain skin samples from the footpad, tail, ear and trunk. We will then label the samples in order to evaluate the presence of DCs (MHCII+CD11c+CD45+) Macrophages (CD11b+F4/80+CD45+), CD8+, and CD4+ T cells.

<table border="0" cellpadding="0" cellspacing="0" width="721">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Reagents</td>
			<td style="width:88px;"></td>
			<td style="width:155px;"></td>
			<td style="width:276px;"></td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:203px;">Supplemented RPMI 1640 medium&#160;</td>
			<td></td>
			<td></td>
			<td style="width:276px;">RPMI1640 supplemented w/ L-glu, pen-strep w/o beta mercapto ethanol</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">FBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Liberase TM (Enzyme Mix)</td>
			<td>Roche</td>
			<td>5401119001</td>
			<td style="width:276px;">resuspend [2.5mg/ml] in dH20 store aliquots at -20&#176;C&#160;</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Dnase I</td>
			<td>Sigma&#160;</td>
			<td>D4263-1VL</td>
			<td style="width:276px;">resuspend in RPMI 1640 w/o serum [1000u/ml]</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Surgical Scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Surgical Forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">35mm petri dishes&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">66mm petri dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">100mm petri dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">75um Cell Strainers</td>
			<td>BD</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company</td>
			<td>Clone</td>
			<td>Label</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">aCD45.2</td>
			<td></td>
			<td>104</td>
			<td>PE-Cy5.5</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">aCD11c</td>
			<td></td>
			<td>N418</td>
			<td>PE-Cy7</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">CD11b</td>
			<td></td>
			<td>M1/70</td>
			<td>FITC</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">F4/80</td>
			<td></td>
			<td>A 3-1</td>
			<td>APC-Cy7</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">aCD4</td>
			<td></td>
			<td>Gk1.5</td>
			<td>APC-Cy7</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">aCD8</td>
			<td></td>
			<td>53-6.7</td>
			<td>PE</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">aCD64</td>
			<td></td>
			<td>X54-5/7.1</td>
			<td>PE-Cy7</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">aCD207</td>
			<td></td>
			<td>4C7</td>
			<td>APC</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Digestion cocktail</td>
			<td></td>
			<td></td>
			<td>Dilute in RPMI 5%FBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">LIberase TM&#160;</td>
			<td></td>
			<td></td>
			<td>300 ug/ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">DNAse1&#160;</td>
			<td></td>
			<td style="width:155px;"></td>
			<td>50 U/ml</td>
		</tr>
	</tbody>
</table>

preparation of single-cell suspensions for cytofluorimetric analysis from different mouse skin regions

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and epidermis home a variety of immune cells in both homeostatic and inflammatory conditions. Tools to obtain skin cell release for cytofluorimetric analyses are, therefore, very useful in order to study the complex network of immune cells residing in the skin and their response to microbial stimuli. Here, we describe an efficient methodology for the digestion of mouse skin to rapidly and efficiently obtain single-cell suspensions. This protocol allows maintenance of maximum cell viability without compromising surface antigen expression. We also describe how to take and digest skin samples from different anatomical locations, such as the ear, trunk, tail, and footpad. The obtained suspensions are then stained and analyzed by flow cytometry to discriminate between different leukocyte populations.

La piel de diferente región del cuerpo se digirió de acuerdo con el protocolo descrito y se tiñeron con los anticuerpos indicados. La estrategia gating se representa en la Figura 1. En primer lugar seleccionar células vivas (células DAPI-), entonces la puerta en singletes (FSC-A vs. FSC-W) y en células con morfología de linfocitos (FSC frente a SSC-A-A). Cuando esté indicado, se seleccionan las células CD45 + de origen hematopoyético. DCs están etiquetados con anti-CD11c, -MHCII, y -CD45 anticuerpos, los macrófagos se identifican como F4 / 80 + CD64 + CD45 + células, células B como CD19 + CD45 + MHCII + células, los linfocitos T CD4 + como CD4 + CD45 + células, y los linfocitos T CD8 + como las células CD8 + CD45 + (Figura 2). Este método guarantees alta viabilidad de la población digerido con más de 50% de células viables totales (DAPI-) en todas las regiones de la piel que son analizados (Figura 3A). Una indicación de la cantidad de células CD45 + rendimiento por cm 2 se representa en la Figura 3B. Una estimación del número total de DCs, los macrófagos, las células B y las células CD4 + o células T CD8 + obtenible a partir de la digestión de 1 cm 2 de piel de ratón se indica en la Figura 3C. <img alt="Figura 1" src="/files/ftp_upload/52589/52589fig1.jpg" /> Figura 1. Identificación de células de origen hematopoyético en la piel suspensiones de una sola célula. Suspensiones unicelulares han sido teñidas con DAPI y el anticuerpo anti-CD45. Entre las células vivas (DAPI-negativo), singletes (FSC-A frente a FSC-H) se analizan para seleccionar células con leucocitos<html> morfología (FSC frente a SSC-A-A). Las células de origen hematopoyético se identifican como CD45 +. <img alt="Figura 2" src="/files/ftp_upload/52589/52589fig2.jpg" /> Figura 2. Análisis de células inmunes presentes en la piel de ratón derivados de diferentes regiones. Las células se cerrada como se indica en la Figura 1. DCs se identifican como CD11c + células MHC II + CD45 + y luego se tiñó con CD207 para distinguir las células de Langerhans y CD207 + dérmica países en desarrollo de CD207 -; Los macrófagos son identificados como CD64 + 80 + CD45 + células / F4; Los linfocitos T se dividen en CD8 + CD45 + células CD4 + y CD45 + en las células; Las células B se identifican como células MHCII + CD19 + CD45 +. Los porcentajes se refieren al total de células CD45 +. &quot;&gt; <img alt="figura 3" src="/files/ftp_upload/52589/52589fig3.jpg" /> Figura 3. Análisis cuantitativo de las células inmunes presentes en la piel de ratón de diferentes regiones (A) Porcentaje de viable. (DAPI -) células derivadas de las regiones indicadas de la piel del ratón. (B) Los números absolutos de células CD45 + en las diferentes regiones de la piel del ratón expresan en número de células por cm2 de piel. (C) Número estimado de DCs, MΦ, las células B y las células CD4 + o células T CD8 + derivados de 1 cm 2 de la piel de las regiones indicadas de la piel del ratón. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Anticuerpo </td><td> tinción </td><td> Concentración </td><td> Hora </td><td> Temperatura </td></tr><tr><td colspan="5"> DCMezcla </td></tr><tr><td> CD11c </td><td> PE-Cy7 </td><td> 2 g / ml </td><td> 30 minutos </td><td> 4 ° C </td></tr><tr><td> MHC II </td><td> EDUCACIÓN FÍSICA </td><td> &quot; </td><td> &quot; </td><td> &quot; </td></tr><tr><td> CD45 </td><td> PE-Cy5.5 </td><td> &quot; </td><td> &quot; </td><td> &quot; </td></tr><tr><td> CD207 </td><td> APC </td><td> &quot; </td><td> &quot; </td><td> &quot; </td></tr><tr><td colspan="5"> Los macrófagos Mix </td></tr><tr><td> F4 / 80 </td><td> APC-Cy7 </td><td> 2 g / ml </td><td> 30 minutos </td><td> 4 ° C </td></tr><tr><td> MHC II </td><td> EDUCACIÓN FÍSICA </td><td> &quot; </td><td> &quot; </td><td> &quot; </td></tr><tr><td> CD45 </td><td> PE-Cy5.5 </td><td> &quot; </td><td> &quot; </td><td> &quot; </td></tr><tr><td> CD64 </td><td> PE-Cy7 </td><td> &quot; </td><td> &quot; </td><td> &quot; </td></tr><tr><td colspan="5"> Mezclar las células T y B </td></tr><tr&gt; <td> CD8a </td><td> EDUCACIÓN FÍSICA </td><td> 2 g / ml </td><td> 30 minutos </td><td> 4 ° C </td></tr><tr><td> CD4 </td><td> APC-Cy7 </td><td> &quot; </td><td> &quot; </td><td> &quot; </td></tr><tr><td> CD19 </td><td> APC </td><td> &quot; </td><td> &quot; </td><td> &quot; </td></tr><tr><td> CD45 </td><td> PE-Cy5.5 </td><td> &quot; </td><td> &quot; </td><td> &quot; </td></tr></tbody></table> Tabla 1. Indicaciones para la tinción de las diferentes poblaciones de células inmunes que pueblan la piel. Tres mezclas se indican para la tinción de las DC, macrófagos y linfocitos.

Watch this Scientific Journal Video about Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions at JoVE.com

Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

The skin is home to a complex immune cell network. We describe an efficient methodology for the digestion of mouse skin, from different parts of the animal's body, in order to obtain a single-cell suspension and analyze the different leukocyte populations resident in the skin by flow cytometry.

Preparación de suspensiones de una sola célula para el Análisis cytofluorimetric de diferentes regiones de piel de ratón

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and ...

preparation-single-cell-suspensions-for-cytofluorimetric-analysis

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

18.2K vistas.  University of Milano-Bicocca. The skin is home to a complex immune cell network. We describe an efficient methodology for the digestion of mouse skin, from different parts of the animal's body, in order to obtain a single-cell suspension and analyze the different leukocyte populations resident in the skin by flow cytometry. 

Video: Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

Preparación de inmunógeno ratón hipófisis para la inducción de la hipofisitis autoinmune experimental

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

Investigación

Inmunología e Infección

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

Preparación de ADN viral de nucleocápside

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

Sola célula de análisis de Bacillus subtilis Las biopelículas mediante microscopía de fluorescencia y citometría de flujo

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Granulocyte-dependent Autoantibody-induced Skin Blistering

Autoimmune phenomena occur in healthy individuals, but when self-tolerance fails, the autoimmune response may result in specific pathology. According to Witebsky's postulates, one of the criteria in diagnosing a disease as autoimmune is the reproduction of the disease in experimental animals by the passive transfer of autoantibodies. For epidermolysis bullosa acquisita (EBA), a prototypic organ-specific autoimmune disease of skin and mucous membranes, several experimental models were recently established. In the animal model described in our present work, purified IgG antibodies against a stretch of 200 amino acids (aa 757-967) of collagen VII are injected repeatedly into mice reproducing the blistering phenotype as well as the histo- and immunopathological features characteristic to human EBA 1. Full-blown widespread disease is usually seen 5-6 days after the first injection and the extent of the disease correlates with the dose of the administered collagen VII-specific IgG. The tissue damage (blister formation) in the experimental EBA is depending on the recruitment and activation of granulocytes by tissue-bound autoantibodies 2,-4. Therefore, this model allows for the dissection of the granulocyte-dependent inflammatory pathway involved in the autoimmune tissue damage, as the model reproduces only the T cell-independent phase of the efferent autoimmune response. Furthermore, its value is underlined by a number of studies demonstrating the blister-inducing potential of autoantibodies in vivo and investigating the mechanism of the blister formation in EBA 1,3,-6. Finally, this model will greatly facilitate the development of new anti-inflammatory therapies in autoantibody-induced diseases. Overall, the passive transfer animal model of EBA is an accessible and instructive disease model and will help researchers to analyze not only EBA pathogenesis but to answer fundamental biologically and clinically essential autoimmunity questions.

In the animal model described in our present work, purified IgG antibodies against a stretch of 200 amino acids (aa 757-967) of collagen VII are injected repeatedly into mice reproducing the blistering phenotype as well as the histo- and immunopathological features characteristic to human epidermolysis bullosa acquisita (EBA)1.

Granulocitos dependiente de autoanticuerpos inducidos por ampollas en la piel

Autoimmune phenomena occur in healthy individuals, but when self-tolerance fails, the autoimmune response may result in specific pathology. According to ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cuantitativo de alto rendimiento de una sola célula de ensayo de citotoxicidad de las células T

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

El aislamiento de linfocitos de la piel humana para el Análisis y fenotípica<em&gt; Ex Vivo</em&gt; Cultivo Celular

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Aislamiento de leucocitos infiltrantes de ratón Piel Usando enzimática Digest y Separación de degradado

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

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 ...

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis allow the spatial localization and visualization of gangliosides involved in CD4+ T cell activation, that complement expression profiling experiments such as flow cytometry, western blotting or real-time PCR. The quantification of ganglioside expression through flow cytometry and their cellular localization through microscopy can be obtained by the use of anti-ganglioside antibodies with high affinity and specificity. Nonetheless, an adequate handling of cells in suspension involves the treatment of culture plates to promote the necessary adherence required for fluorescence or confocal microscopy acquisition. In this work, we describe a protocol for determining GD3 and GD2 ganglioside expression and colocalization with the TCR during na&#239;ve CD4+ T cell activation. Also, real-time PCR experiments using &#60;40,000 cells are described for the determination of the GD3 and GM2/GD2 synthase genes, demonstrating that gene analysis experiments can be performed with a low number of cells and without the need of additional low input RNA kits.

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

We describe a standard antibody staining protocol for use in microscopy to determine the membrane expression and localization of gangliosides in resting and activated human na&#239;ve CD4+ T cells. Also described are real-time PCR experiments using &#60;40,000 cells that do not require additional low input RNA kits.

Preparación de CD4<sup&gt; +</sup&gt; Las células T para el Análisis de GD3 y GD2 gangliósidos membrana Expresión mediante microscopía

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

Análisis unicelular de Immunophenotype y producción de citoquinas en sangre periférica por citometría de masas

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

Preparación de suspensiones de una sola célula para el Análisis cytofluorimetric de diferentes regiones de piel de ratón

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