Nos gustaría dar las gracias a las subvenciones del NIH para la financiación R01-AI026328 (KAK) y R01-AI079004 (KAK).

1. La eliminación del tracto genital <ol><li> Sacrificar al ratón con las directrices aprobadas. Hacer incisión media baja y retraer la piel. </li><li> Aislar y retirar tracto genital completa, desde el oviducto de la apertura vaginal. </li><li> Abrir todo el tracto longitudinalmente con unas tijeras. </li><li> Cortar el tracto genital con tijeras quirúrgicas finas en piezas finas (aproximadamente &lt;1 mm) en una placa de Petri con RPMI-10/EDTA completa (véase la fórmula a continuación) y se agita el tejido en un matraz de 125 ml a temperatura ambiente en un agitador magnético ajustado para 15 min. Este procedimiento se repite dos veces y luego el sobrenadante, que contiene epitelio y otros residuos, se descarta. </li><li> Verter el contenido del frasco a través de un filtro de células (40μm). Limpie el residuo con EDTA medio completo. Completa RPMI/10: 500 ml RPMI (Gibco), 10% suero bovino fetal (FBS), inactivado por calor, 5 ml de penicilina / estreptomicina, 5 ml de HEPES 1M. RPMI-10/EDTA completa: 10% de RPMI (como anteriormente) con E 5μMDTA </li></ol> 2. La digestión de los tejidos del tracto genital <ol><li> Transferir las piezas de tejido a un nuevo matraz con 20 ml RPMI-10/collagenase fresco (véase la receta a continuación) y digerir el tejido a 37 º C durante 1 hora en un agitador magnético ajustado a agitar vigorosamente el tejido. Completa RPMI-10/collagenase: Justo antes de usar, reconstituir colagenasa tipo VIII (Sigma; almacenar a -20 ° C, o de acuerdo con las instrucciones del fabricante.), Añadir en RPMI/10 completa a una concentración final de 450-500U/ml. </li><li> Después de la primera digestión, recoger los sobrenadantes a través de filtro de células 100 micras, mantener las células en hielo. </li><li> Transferir el tejido no digerido a un nuevo matraz y repetir el paso 2,1 dos veces más utilizando RPMI-10/collagenase completo fresco para un total de 3 digestiones separadas. En este punto, no visibles trozos de tejido debe estar en solución. </li><li> Reunir los sobrenadantes todos juntos de una muestra y recoger a través de filtro de células 100 micras. Haga girar la cElls abajo. Los sedimentos celulares se separaron usando gradientes de Percoll. </li></ol> 3. La separación de las células del tracto genital utilizando gradientes de Percoll <ol><li> Preparar cada concentración de Percoll antes de su uso: <ul><li> 100% Percoll isotónico: mix 09:01 existencias Percoll (Pharmacia Biotch) vs 10x PBS. pH a 7,4 con HCl. </li><li> 40% de Percoll solución: mezcla de 40 ml de Percoll isotónico 100% y 60 ml de RPMI completo. </li><li> 70% de Percoll solución: mezcla de 70 ml de 100% de Percoll isotónico con 30 ml de RPMI completo. </li></ul></li><li> Resuspender cada sedimento de células de la etapa 6 en soluciones de Percoll 40%. Un volumen igual de 70% Percoll se usa para hacer los gradientes. Hay dos formas de configurar los tubos de gradiente: <ol><li> Bajo la capa de: añadir la suspensión celular con 40% de Percoll en un tubo de gradiente de primera, a continuación, cuidadosamente bajo la capa de Percoll 70%. </li><li> El exceso de capa: añadir 70% de Percoll en un tubo primero, entonces lentamente y con cuidado cargar 40% de Percoll que contiene las células en laparte superior. </li></ol></li><li> Las muestras se centrifugaron gradiente @ 900 xg, a temperatura ambiente, durante 20 minutos con el freno desconectado. </li><li> Los linfocitos será en la capa de interfase entre 40% y 70% de Percoll capas. Con cuidado, cosecha de la capa de interfaz, lavar con RPMI 1:3 y centrifugar a 740 xg. Los linfocitos estará en el sedimento celular y están listos para su uso posterior. </li></ol> 4. Los resultados representativos Un ejemplo de aislamiento de linfocitos de ratón tracto genital y el análisis de citometría de flujo (FACS) se muestra en la figura 1. Tracto genital fue retirado de Chlamydia muridarum intravaginal ratón infectado 7 días después de la infección. Dos vías genitales se agruparon para tener suficientes linfocitos para el examen funcional y fenotipo por FACS. Tejidos disecados fueron procesados ​​por la digestión y etapas de aislamiento como se indica anteriormente. La suspensión de células individuales se tiñeron para diversos conjugado a un fluorocromoanticuerpos monoclonales contra CD3 de ratón, CD4 y CD8. La Figura 1 muestra una presentación punto de parcelas de células T CD4 positivas frente a células T CD8 positivas CD3 cerrada en linfocitos T positivos. Nuestro procedimiento puede examinar los linfocitos aislados de los tractos genitales de los diferentes modelos de la enfermedad para evaluar diversas características de las células inmunes funcionales y fenotípicas en diferentes enfermedades en modelo de ratón. Estos incluyen diversas células del sistema inmune, tales como células dendríticas, neutrófilos, macrófagos NK, NKT, células T (Ag específico de células T), células B 3-10 etc. <img alt="Figura 1" src="/files/ftp_upload/4391/4391fig1.jpg" /> Los linfocitos Figura 1. Fueron aisladas de las vías genitales de ratones infectados con Chlamydia muridarum, utilizando el método presentado aquí. Después de que el procedimiento de separación completa, los linfocitos se tiñeron con conjugados con fluorocromo CD3, CD4 y CD8. Los datos fueron cuadrante seleccionado para linfocitos CD3 +,mostrando CD4 + CD8 + frente y con el porcentaje de células cerradas.

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	<li>M, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+perinatal+immune+development+on+mucosal+homeostasis+and+chronic+inflammation.">The impact of perinatal immune development on mucosal homeostasis and chronic inflammation.</a> Nat. Rev Immunol. 12, 9-23 (2011).</li><li>Centers for Disease Control and Prevention. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 2010 Sexually Transmitted Diseases Surveillance. , (2010).</li><li>Jain, S., Patrick, A. J., Rosenthal, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+tandem+copies+of+conserved+gp41+epitopes+incorporated+in+gag+virus-like+particles+elicit+systemic+and+mucosal+antibodies+in+an+optimized+heterologous+vector+delivery+regimen.">Multiple tandem copies of conserved gp41 epitopes incorporated in gag virus-like particles elicit systemic and mucosal antibodies in an optimized heterologous vector delivery regimen.</a> Vaccine. 28, 7070-7080 (2010).</li><li>Tang, V. A., Rosenthal, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravaginal+infection+with+herpes+simplex+virus+type-2+(HSV-2)+generates+a+functional+effector+memory+T+cell+population+that+persists+in+the+murine+genital+tract.">Intravaginal infection with herpes simplex virus type-2 (HSV-2) generates a functional effector memory T cell population that persists in the murine genital tract.</a> J. Reprod. Immunol. 87, 39-44 (2010).</li><li>Gillgrass, A. E., Tang, V. A., Towarnicki, K. M., Rosenthal, K. L., Kaushic, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+against+genital+herpes+infection+in+mice+immunized+under+different+hormonal+conditions+correlates+with+induction+of+vagina-associated+lymphoid+tissue.">Protection against genital herpes infection in mice immunized under different hormonal conditions correlates with induction of vagina-associated lymphoid tissue.</a> J. Virol. 79, 3117-3126 (2005).</li><li>Sajic, D., Patrick, A. J., Rosenthal, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+delivery+of+CpG+oligodeoxynucleotides+expands+functional+dendritic+cells+and+macrophages+in+the+vagina.">Mucosal delivery of CpG oligodeoxynucleotides expands functional dendritic cells and macrophages in the vagina.</a> Immunology. 114, 213-224 (2005).</li><li>Jiang, J. Q., Patrick, A., Moss, R. B., Rosenthal, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8++T-cell-mediated+cross-clade+protection+in+the+genital+tract+following+intranasal+immunization+with+inactivated+human+immunodeficiency+virus+antigen+plus+CpG+oligodeoxynucleotides.">CD8+ T-cell-mediated cross-clade protection in the genital tract following intranasal immunization with inactivated human immunodeficiency virus antigen plus CpG oligodeoxynucleotides.</a> J. Virol. 79, 393-400 (2005).</li><li>Gill, N., Chenoweth, M. J., Verdu, E. F., Ashkar, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NK+cells+require+type+I+IFN+receptor+for+antiviral+responses+during+genital+HSV-2+infection.">NK cells require type I IFN receptor for antiviral responses during genital HSV-2 infection.</a> Cell Immunol. 269, 29-37 (2011).</li><li>Thatte, A., DeWitte-Orr, S. J., Lichty, B., Mossman, K. L., Ashkar, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+for+IL-15+in+TLR-mediated+innate+antiviral+immunity+against+genital+HSV-2+infection.">A critical role for IL-15 in TLR-mediated innate antiviral immunity against genital HSV-2 infection.</a> Immunol. Cell Biol. 89, 663-669 (2011).</li><li>Kwant-Mitchell, A., Ashkar, A. A., Rosenthal, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+innate+and+adaptive+immune+responses+against+herpes+simplex+virus+type+2+in+a+humanized+mouse+model.">Mucosal innate and adaptive immune responses against herpes simplex virus type 2 in a humanized mouse model.</a> J. Virol. 83, 10664-10676 (2009).</li><li>Gallichan, W. S., Rosenthal, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+estrous+cycle+on+local+humoral+immune+responses+and+protection+of+intranasally+immunized+female+nice+against+herpes+simplex+virus+type+2+infection+in+the+genital+tract.">Effects of the estrous cycle on local humoral immune responses and protection of intranasally immunized female nice against herpes simplex virus type 2 infection in the genital tract.</a> Virology. , 224-487 (1996).</li></ol>

No hay conflictos de interés declarado.

En este protocolo, se muestra cómo preparar los linfocitos de ratón tractos genitales y se domina con facilidad. El punto más crítico de este proceso es la de mantener la viabilidad celular, manteniendo las células en hielo. El rendimiento celular de este método depende de la técnica, el manejo de la habilidad, el estado infeccioso del ratón (ingenua o días después de la infección), el patógeno (bacteria, virus hongos,) y la sección de la murina examinados tracto reproductivo (oviducto, útero, segmento vag...

<table border="1"><tbody><tr><td> Nombre </td><td> Empresa </td><td> Número de catálogo </td><td></td></tr><tr><td> La colagenasa </td><td> Sigma Ciencias de la Vida </td><td> C2139-1G </td><td></td></tr><tr><td> Percoll </td><td> GE Healthcare Bio-Sciences </td><td> 17-0891-01 </td><td></td></tr><tr><td> RPMI1640 </td><td> Por Gibco Life Technologies </td><td> 11875 </td><td></td></tr></tbody></table>

isolation of lymphocytes from mouse genital tract mucosa

Las superficies mucosas, incluso en los tractos gastrointestinales, urogenitales y respiratorias, ofrecen puertas de entrada para los patógenos, como los virus y las bacterias 1. Mucosas también son sitios inductivos en el huésped para generar inmunidad contra los agentes patógenos, tales como las placas de Peyer en el tracto intestinal y el tejido linforreticular nasal asociada en el tracto respiratorio. Esta característica única hace inmunidad de la mucosa como actor fundamental del sistema de defensa del huésped. Muchos estudios se han centrado en los sitios de la mucosa gastrointestinal y respiratorio. Sin embargo, ha habido muy poca investigación de la reproducción sitios de la mucosa. La mucosa del tracto genital es el sitio de la infección primaria de las enfermedades de transmisión sexual (ETS), incluyendo infecciones bacterianas y virales. Las ETS son uno de los retos de salud más críticos que enfrenta el mundo hoy en día. Centros para el Control y Prevención de Enfermedades estima que hay 19 millones de nuevos infecciosas cada año en los Estados Unidos. STDs costar la salud de los EE.UU. sistema de atención de 17 mil millones dólares cada año 2, y los individuos de costes aún más en las consecuencias inmediatas de salud y larga vida. Para hacer frente a este reto, una mayor comprensión de la inmunidad de la mucosa reproductiva es necesaria y aislar linfocitos es un componente esencial de estos estudios. Aquí, se presenta un método para aislar reproducible linfocitos murinos de tractos genitales femeninos por estudios inmunológicos que pueden ser modificadas para la adaptación a otras especies. El método se describe a continuación se basa en un ratón.

Watch this Scientific Journal Video about Isolation of Lymphocytes from Mouse Genital Tract Mucosa at JoVE.com

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Un modo eficaz de aislar los linfocitos de ratón tracto genital se describe. Este método tiene la ventaja de digestión enzimática y separación por gradiente de Percoll para permitir el aislamiento eficiente. Esta técnica es también adaptable a para su uso en otras especies

Aislamiento de linfocitos de ratón del tracto genital Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

isolation-of-lymphocytes-from-mouse-genital-tract-mucosa

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Immunology and Infection

14.3K vistas.  University of California, Los Angeles. Un modo eficaz de aislar los linfocitos de ratón tracto genital se describe. Este método tiene la ventaja de digestión enzimática y separación por gradiente de Percoll para permitir el aislamiento eficiente. Esta técnica es también adaptable a para su uso en otras especies

Video: Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Transurethral Induction of Mouse Urinary Tract Infection

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on Luria-Bertani (LB) agar at 37&deg;C in ambient air. UPEC strains grow as yellowish-white translucent colonies on LB agar. Following confirmation of appropriate colony morphology, single colonies are then picked to be cultured in broth. LB broth can be used for most uropathogenic bacterial strains. Two serial, overnight LB broth cultures can be employed to enhance expression of type I pili, a well-defined virulence factor for uropathogenic bacteria. Broth cultures are diluted to the desired concentration in phosphate buffered saline (PBS). Eight to 12 week old female mice are placed under isoflurane anesthesia and transurethrally inoculated with bacteria using polyethylene tubing-covered 30 gauge syringes. Typical inocula, which must be empirically determined for each bacterial/mouse strain combination, are 106 to 108 cfu per mouse in 10 to 50 microliters of PBS. After the desired infection period (one day to several weeks), urine samples and the bladder and both kidneys are harvested. Each organ is minced, placed in PBS, and homogenized in a Blue Bullet homogenizer. Urine and tissue homogenates are serially diluted in PBS and cultured on appropriate agar. The following day, colony forming units are counted.

This video will demonstrate methods to transurethrally induce mouse urinary tract infections and quantify the extent of resulting infections.

Inducción transuretral de infección del tracto urinario ratón

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on ...

Investigación

Inmunología e Infección

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

El aislamiento de ARN Pseudomonas aeruginosa Que colonizan el tracto gastrointestinal murino

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

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

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

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 and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Aislamiento y diferenciación de Th17 Naïve linfocitos T CD4

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Análisis rápido de aberraciones cromosómicas en linfocitos B de ratón por PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Protocolo Aislamiento eficiente para B y linfocitos T a partir Humanos Palatine amígdalas

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

El aislamiento y la citometría de flujo Caracterización de murino Small intestinal linfocitos

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Aislamiento y activación de los linfocitos murinos

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

Ex Vivo Infección de la Mucosa Genital tejido linfoide humano y mujer con Virus de inmunodeficiencia humana 1 y Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

Aislamiento de las células dendríticas del tracto reproductivo femenino humano estudios fenotípica y funcional

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...