Nous tenons à remercier NIH pour les subventions de financement R01-AI026328 (KAK) et R01-AI079004 (KAK).

1. Retrait de l&#39;appareil génital <ol><li> Euthanasier la souris en utilisant les lignes directrices approuvées. Faire une incision médiane bas et retirer la peau. </li><li> Isoler et supprimer tout appareil génital, de l&#39;oviducte dans l&#39;ouverture du vagin. </li><li> Ouvrez l&#39;ensemble du tractus longitudinalement avec des ciseaux. </li><li> Couper les voies génitales avec de beaux ciseaux chirurgicaux en petits morceaux (environ &lt;1mm) dans une boîte de Pétri avec RPMI-10/EDTA complète (voir la formule ci-dessous) et agiter le tissu dans 125 ml flacon à température ambiante sur un agitateur magnétique réglé pour 15 min. Cette procédure est répétée deux fois, puis le surnageant, qui contient l&#39;épithélium et d&#39;autres débris, est rejetée. </li><li> Verser le contenu du flacon à travers un tamis cellulaire (40 um). Enlever tout résidu de l&#39;EDTA avec du milieu complet. RPMI/10 complète: 500 ml de RPMI (Gibco), 10% de sérum bovin foetal (FBS), inactivé à la chaleur, 5 ml de pénicilline / streptomycine, 5 ml de HEPES 1M. Complète RPMI-10/EDTA: RPMI 10% (comme ci-dessus) avec E 5 uMDTA </li></ol> 2. La digestion des tissus des voies génitales <ol><li> Transfert morceaux de tissu pour un nouveau flacon de 20 ml RPMI-10/collagenase frais (voir la recette ci-dessous) et de digérer les tissus à 37 ° C pendant 1 heure sur un agitateur magnétique réglé à remuer vigoureusement le tissu. RPMI-10/collagenase complet: Juste avant utilisation, reconstituer la collagénase de type VIII (Sigma, conserver à -20 ° C, ou selon les instructions du fabricant.), Ajouter dans RPMI/10 complète à une concentration finale de 450-500U/ml. </li><li> Après la première digestion, de recueillir les surnageants à travers un tamis de 100 um cellule, maintenir les cellules sur la glace. </li><li> Transférer le tissu non digérés dans un ballon à nouveau et répétez l&#39;étape 2.1 deux fois à l&#39;aide de frais RPMI-10/collagenase complet pour un total de 3 digestions distinctes. À ce stade, aucun morceaux de tissu doivent être visibles dans la solution. </li><li> Réunir les fractions surnageantes tous ensemble à partir d&#39;un échantillon et collecter à travers un tamis de 100 um cellule. Faites tourner la cElls vers le bas. Les culots cellulaires sont séparés en utilisant des gradients de Percoll. </li></ol> 3. Séparation des cellules du tractus génital en utilisant des gradients de Percoll <ol><li> Préparer chaque concentration de Percoll avant de l&#39;utiliser: <ul><li> 100% Percoll isotonique: mélange 09:01 de stock Percoll (Pharmacia Biotch) vs 10x PBS. il le pH à 7,4 à l&#39;aide HCL. </li><li> Solution Percoll 40%: mélanger 40 ml de Percoll isotonique 100% et de 60 ml de RPMI complet. </li><li> Solution Percoll 70%: mélanger 70 ml de Percoll isotonique 100% avec 30 ml de RPMI complet. </li></ul></li><li> Remettre en suspension chaque culot cellulaire de l&#39;étape 6 dans les solutions de Percoll 40%. Un volume égal de Percoll 70% sera utilisé pour faire les dégradés. Il ya deux façons de mettre en place des tubes de gradient: <ol><li> Sous-couche: ajouter suspension cellulaire avec Percoll 40% dans un tube à gradient d&#39;abord, puis soigneusement sous-couche de Percoll 70%. </li><li> Sur-couche: ajouter Percoll 70% dans le tube, puis soigneusement et lentement charger Percoll 40% contenant les cellules de ladessus. </li></ol></li><li> Gradient échantillons seront centrifugés à 900 xg température ambiante,, pendant 20 minutes avec le frein hors tension. </li><li> Les lymphocytes seront à la couche d&#39;interface entre 40% et 70% des couches de Percoll. Soigneusement récolter la couche d&#39;interface, laver avec du RPMI 1:3 et spin bas à 740 xg. Les lymphocytes sont dans le culot cellulaire et sont prêts pour une utilisation ultérieure. </li></ol> 4. Les résultats représentatifs Un exemple de l&#39;isolement des lymphocytes de souris tractus génital et cytométrie de flux (FACS) est illustré à la figure 1. Tractus génital a été retiré de Chlamydia muridarum intravaginale souris infectées 7 jours après l&#39;infection. Deux voies génitales ont été regroupées afin d&#39;avoir suffisamment de lymphocytes pour examen fonctionnel et le phénotype par FACS. Tissus disséqués ont été traitées par la digestion et étapes d&#39;isolation tel que décrit ci-dessus. La suspension cellulaire unique a été coloré pour diverses conjugué à un fluorochromeanticorps monoclonaux dirigés contre CD3 de souris, CD4 et CD8. La figure 1 montre une présentation dot-plot des cellules T CD4 positifs par rapport aux lymphocytes T CD8 positifs gated sur CD3 des lymphocytes T positifs. Notre procédure peut examiner lymphocytes isolés à partir des voies génitales de modèles de maladies différentes pour évaluer les différentes caractéristiques de cellules immunitaires fonctionnelles et phénotypiques dans différentes maladies dans le modèle de souris. Il s&#39;agit notamment de diverses cellules immunitaires, telles que les cellules dendritiques, les macrophages, polynucléaires neutrophiles NK, NKT, les lymphocytes T (Ag cellules T spécifiques), les lymphocytes B, etc 3-10. <img alt="Figure 1" src="/files/ftp_upload/4391/4391fig1.jpg" /> Lymphocytes Figure 1. Ont été isolés à partir des voies génitales de souris infectées par la chlamydia muridarum, en utilisant la méthode présentée ici. Après la procédure de séparation complète, les lymphocytes ont été colorés avec un fluorochrome conjugués CD3, CD4 et CD8. Les données ont été quadrant gated sur lymphocytes CD3 +,montrant CD4 + CD8 + par rapport aux cellules et le pourcentage de dépendants.

<ol>
	<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>

Aucun conflit d&#39;intérêt déclaré.

Dans ce protocole, il est montré comment préparer des lymphocytes de souris voies génitales et est facilement maîtrisé. Le point le plus critique de ce processus est de maintenir la viabilité des cellules en gardant les cellules sur la glace. Le rendement cellulaire de cette méthode dépend de la technique, la gestion des compétences, le statut infectieux de la souris (ou naïve jour après l&#39;infection), l&#39;agent pathogène (bactérie, virus, champignon) et la section de la souris examiné l&#39;appareil ...

<table border="1"><tbody><tr><td> Nom </td><td> Entreprise </td><td> Numéro de catalogue </td><td></td></tr><tr><td> Collagénase </td><td> Sigma Sciences de la Vie </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> Gibco par Life Technologies </td><td> 11875 </td><td></td></tr></tbody></table>

isolation of lymphocytes from mouse genital tract mucosa

Les muqueuses, y compris dans les voies gastro-intestinales, génito-urinaire, respiratoire et, fournir des portes d&#39;entrée pour les agents pathogènes, comme les virus et les bactéries 1. Muqueuses sont également des sites inductifs dans l&#39;hôte afin de générer une immunité contre les agents pathogènes, tels que les patchs Peyers dans le tractus intestinal et le tissu nasal lymphoréticulaire associé dans les voies respiratoires. Cette caractéristique unique permet de l&#39;immunité des muqueuses comme un acteur incontournable du système de défense de l&#39;hôte. De nombreuses études ont porté sur digestives et respiratoires dans les muqueuses. Cependant, il ya eu peu d&#39;études de la reproduction des muqueuses. La muqueuse du tractus génital est le site d&#39;infection primaire des maladies sexuellement transmissibles (MST), y compris les infections bactériennes et virales. MST sont un des problèmes de santé les plus critiques auxquels est confronté le monde d&#39;aujourd&#39;hui. Centers for Disease Control and Prevention estime qu&#39;il ya 19 millions de nouveaux infectieuse chaque année aux États-Unis. STDs le coût du système de santé américain 17 milliards de dollars chaque année 2, et les individus de coûts encore plus en conséquences immédiates et à long de la vie. Pour faire face à ce défi, une meilleure compréhension de l&#39;immunité muqueuse reproduction est nécessaire et isoler des lymphocytes est une composante essentielle de ces études. Ici, nous présentons une méthode reproductible pour isoler les lymphocytes murins de voies génitales femelles pour les études immunologiques qui peuvent être modifiés pour s&#39;adapter à d&#39;autres espèces. La méthode décrite ci-après est basé sur une souris.

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 moyen efficace pour isoler les lymphocytes de souris tractus génital est décrite. Cette méthode tire parti de la digestion enzymatique et séparation par gradient de Percoll pour permettre l&#39;isolement efficace. Cette technique est également adaptable pour une utilisation à d&#39;autres espèces

Isolement des lymphocytes de souris muqueuses génitales Tract

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 vues.  University of California, Los Angeles. Un moyen efficace pour isoler les lymphocytes de souris tractus génital est décrite. Cette méthode tire parti de la digestion enzymatique et séparation par gradient de Percoll pour permettre l'isolement efficace. Cette technique est également adaptable pour une utilisation à d'autres espèces

Vidéo: 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.

Induction transurétrale d&#39;infection des voies urinaires souris

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

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Immunologie et infection

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.

Isolement des ARN Pseudomonas aeruginosa La colonisation du tractus gastro-intestinal murin

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

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer&#039;s Patches

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

Et isoler immunocoloration des lymphocytes et des cellules dendritiques à partir des plaques de Peyer murin de

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance 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&amp;#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.

L&#39;isolement et la différenciation Th17 de Naïve CD4 des lymphocytes T

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.

Analyse rapide des aberrations chromosomiques dans les lymphocytes B de souris par 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.

Protocole d&#39;isolation efficace pour B et les lymphocytes T à partir des amygdales palatines humaines

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.

L&#39;isolement et la caractérisation par cytométrie en flux de l&#39;intestin grêle Lymphocytes murine

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.

Isolement et activation de murins Lymphocytes

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 Infection de la muqueuse génitale humain tissu lymphoïde et femelle avec le Virus de l’immunodéficience humaine 1 et 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.

Isolement de cellules dendritiques de l’appareil reproducteur féminin humain pour des études phénotypiques et fonctionnelles

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