Ci piace ringraziare NIH per le sovvenzioni di finanziamento R01-AI026328 (KAK) e R01-AI079004 (KAK).

1. Rimozione del tratto genitale <ol><li> Eutanasia il mouse secondo le linee guida approvate. Fai incisione bassa linea mediana e ritrarre la pelle. </li><li> Isolare e rimuovere tutto tratto genitale, da ovidotto all&#39;apertura vaginale. </li><li> Aprire l&#39;intero tratto longitudinale con le forbici. </li><li> Tagliare il tratto genitale con belle forbici chirurgiche in pezzi sottili (circa &lt;1 mm) in una capsula di Petri con RPMI-10/EDTA completa (vedere la formula di seguito) e mescolare il tessuto in 125 ml matraccio a temperatura ambiente su un agitatore magnetico fissato per 15 min. Questa procedura viene ripetuta due volte e poi il surnatante, che contiene epitelio e altri detriti, viene scartato. </li><li> Versare il contenuto del matraccio attraverso un colino cellula (40μm). Lavare i residui EDTA con terreno completo. Completa RPMI/10: 500 ml RPMI (Gibco), 10% di siero fetale bovino (FBS), inattivato al calore, 5 ml di penicillina / streptomicina, 5 ml 1M HEPES. Completa RPMI-10/EDTA: 10% RPMI (come sopra) con E 5 micronDTA </li></ol> 2. La digestione dei tessuti del tratto genitale <ol><li> Trasferire pezzi di tessuto in un pallone nuovo con 20 ml RPMI-10/collagenase fresco (vedi ricetta sotto) e digerire il tessuto a 37 ° C per 1 ora su un agitatore magnetico impostato agitare energicamente il tessuto. Completa RPMI-10/collagenase: Giusto prima dell&#39;uso, ricostituire collagenasi di tipo VIII (Sigma; conservare a -20 ° C, o secondo le istruzioni del produttore.), Aggiungere nel RPMI/10 completa ad una concentrazione finale di 450-500U/ml. </li><li> Dopo la prima digestione, raccogliere surnatanti con filtro da 100 micron delle cellule, mantenere le cellule in ghiaccio. </li><li> Trasferire il tessuto non digerito in un pallone nuovo e ripetere il punto 2,1 per altre due volte utilizzando fresco RPMI-10/collagenase completo per un totale di 3 digestioni separate. A questo punto, non visibili pezzi di tessuto devono essere in soluzione. </li><li> Piscina tutti i surnatanti insieme da un campione e raccogliere con filtro da 100 micron cella. Gira la cElls verso il basso. Pellet cellulari saranno separati utilizzando gradienti Percoll. </li></ol> 3. Separare le cellule del tratto genitale con gradienti Percoll <ol><li> Preparare ciascuna concentrazione di Percoll prima dell&#39;uso: <ul><li> 100% isotonica Percoll: mix 9:01 magazzino Percoll (Pharmacia BiOTCH) vs 10x PBS. pH a 7,4 utilizzando HCl. </li><li> Percoll soluzione al 40%: miscelare 40 ml di Percoll isotonico 100% e 60 ml di RPMI completo. </li><li> Percoll soluzione al 70%: miscelare 70 ml del 100% Percoll isotonico con 30 ml di RPMI completo. </li></ul></li><li> Risospendere ogni pellet di cellule dal punto 6 in soluzioni Percoll 40%. Un volume uguale di Percoll 70% sarà utilizzato per fare i gradienti. Ci sono due modi per impostare i tubi di gradiente: <ol><li> Under-strato: aggiungi sospensione cellulare con il 40% in un tubo Percoll primo gradiente, poi accuratamente sotto-strato Percoll 70%. </li><li> Over-strato: aggiungere 70% Percoll nel tubo, poi lentamente e con cautela carico massimo di 40% Percoll contenente le celle deltop. </li></ol></li><li> Gradiente campioni saranno centrifugati @ 900 xg, temperatura ambiente, per 20 minuti con il freno. </li><li> I linfociti saranno al livello di interfaccia tra il 40% e strati di Percoll 70%. Cautela raccogliere il livello di interfaccia, lavare con RPMI 1:3 e spin down a 740 xg. I linfociti saranno nel pellet cellulare e sono pronti per un ulteriore uso. </li></ol> 4. Risultati rappresentativi Un esempio di isolamento dei linfociti di topo tratto genitale e citometria a flusso (FACS) analisi è illustrato nella figura 1. Tratto genitale è stato rimosso da Chlamydia muridarum intravaginale del mouse infettato 7 giorni dopo l&#39;infezione. Due vie genitali sono stati raggruppati insieme in modo da avere linfociti sufficienti per esame funzionale e fenotipo mediante FACS. Tessuti sezionati sono stati trattati con la digestione e passaggi di isolamento come descritto sopra. La sospensione singola cellula è stato colorato per vari fluorocromo coniugatoanticorpi monoclonali contro CD3 del mouse, CD4 e CD8. La figura 1 mostra una dot-plot presentazione di cellule CD4 positive T contro le cellule T CD8 positivi gate sui linfociti T CD3 positivi. La nostra procedura in grado di esaminare i linfociti isolati da vie genitali di modelli di malattia diversi per valutare diverse caratteristiche funzionali delle cellule immunitarie e fenotipica in malattie diverse in modello murino. Questi includono diverse cellule immunitarie, come le cellule dendritiche, macrofagi, neutrofili NK, NKT, cellule T (Ag-specifiche cellule T), cellule B ecc 3-10. <img alt="Figura 1" src="/files/ftp_upload/4391/4391fig1.jpg" /> Linfociti Figura 1. Sono stati isolati dal tratto genitale di topi infettati con Chlamydia muridarum, utilizzando il metodo qui presentato. Dopo la procedura di separazione completa, i linfociti sono stati colorati con fluorocromi coniugati CD3, CD4 e CD8. I dati erano quadrante gate sui linfociti CD3 +,mostrando CD4 + CD8 + con rispetto e la percentuale di cellule chiuse.

<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>

Nessun conflitto di interessi dichiarati.

In questo protocollo, si mostra come preparare i linfociti da vie genitali del mouse ed è facilmente padronanza. Il punto più critico di questo processo è di mantenere la vitalità cellulare, mantenendo le cellule in ghiaccio. La quantità di cellule da questo metodo dipende dalla tecnica, manipolazione abilità, lo stato infettivo del mouse (ingenua o giorni dopo l&#39;infezione), il patogeno (batterica, virale, fungina) e la sezione del tratto riproduttivo murino esaminati (ovidotto, utero, segmento vaginale o l&#3...

<table border="1"><tbody><tr><td> Nome </td><td> Azienda </td><td> Numero di catalogo </td><td></td></tr><tr><td> Collagenasi </td><td> Sigma Life Sciences </td><td> C2139-1G </td><td></td></tr><tr><td> Percoll </td><td> GE Healthcare Bio-Scienze </td><td> 17-0891-01 </td><td></td></tr><tr><td> RPMI1640 </td><td> Gibco da Life Technologies </td><td> 11875 </td><td></td></tr></tbody></table>

isolation of lymphocytes from mouse genital tract mucosa

Superfici mucose, anche nel tratto gastrointestinale, urogenitale e respiratorio, fornire portali di ingresso per i patogeni, come virus e batteri 1. Mucose sono anche siti induttivi nell&#39;ospite per generare immunità contro patogeni, come i cerotti Peyers nel tratto intestinale e nasale associata tessuto linforeticolare nel tratto respiratorio. Questa caratteristica unica porta immunità mucosale come un giocatore fondamentale del sistema difesa ospite. Molti studi si sono concentrati sui siti della mucosa gastrointestinale e respiratorio. Tuttavia, vi è stata piccola indagine riproduttivi siti mucosali. La mucosa del tratto genitale è il sito di infezione primaria per le malattie sessualmente trasmissibili (MST), comprese le infezioni batteriche e virali. Malattie a trasmissione sessuale sono una delle sfide più critiche per la salute di fronte al mondo di oggi. Centri per il Controllo e la Prevenzione stima che ci sono 19 milioni di nuovi infetti ogni anno negli Stati Uniti. STDs costato il sistema sanitario degli Stati Uniti 17 miliardi dollari ogni anno 2, e gli individui dei costi ancora di più in immediate conseguenze per la salute e per tutta la vita. Per far fronte a questa sfida, una maggiore comprensione di riproduzione immunità mucosale è necessario e isolare i linfociti è una componente essenziale di questi studi. Qui, vi presentiamo un metodo per isolare in modo riproducibile da linfociti murini vie genitali femminili per gli studi immunologici che possono essere modificati per adattarsi ad altre specie. Il metodo descritto di seguito è basato su un mouse.

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 efficace per isolare i linfociti di topo tratto genitale è descritto. Questo metodo si avvale di digestione enzimatica e la separazione gradiente Percoll per consentire l&#39;isolamento efficiente. Questa tecnica è anche adattabile a per l&#39;uso in altre specie

Isolamento dei linfociti da mouse mucosa del tratto genitale

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

Research

JoVE Journal

Immunology and Infection

14.2K Views.  University of California, Los Angeles. Un modo efficace per isolare i linfociti di topo tratto genitale è descritto. Questo metodo si avvale di digestione enzimatica e la separazione gradiente Percoll per consentire l'isolamento efficiente. Questa tecnica è anche adattabile a per l'uso in altre specie

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.

Induzione transuretrale di infezioni del tratto urinario mouse

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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Immunologia e infezioni

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.

Isolamento del RNA Pseudomonas aeruginosa Colonizzare la Murine tratto gastrointestinale

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.

Isolare e Immunostaining linfociti e le cellule dendritiche da placche di Peyer murino di

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.

Isolamento e differenziazione di Th17 Naïve linfociti 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.

Analisi rapida di aberrazioni cromosomiche in linfociti B mouse da 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.

Efficiente protocollo Isolamento da B e T linfociti umani da Palatine Tonsille

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.

Isolamento e caratterizzazione citometria a flusso di Murine Piccolo intestinale Linfociti

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.

L&#39;isolamento e l&#39;attivazione di linfociti murini

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 Infezione della Mucosa genitale umana del tessuto linfoide e donna con il Virus dell'immunodeficienza umana 1 e 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.

Isolamento di cellule dendritiche dal tratto riproduttivo femminile umano per studi fenotipici e funzionali

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