NKS is supported by NIH award K08 AI108690.

Tutti gli studi sono stati condotti in esame rigoroso e linee guida secondo il Comitato Istituzionale Animal Care e Usa (IACUC) presso la Harvard Medical School, che risponde alle norme veterinarie stabilite dalla American Association for Laboratory Animal Science (AALAS). 1. orizzontale trasmissione di batteri attraverso Co-housing (opzionale) <ol><li> Per ridurre al minimo la contaminazione esogena (in particolare se si utilizza topi gnotobiotic), praticare la tecnica asettica, mentre l&#39;assemblaggio gabbie sterili monouso, con cibo e acqua che sono stati entrambi in autoclave. </li><li> Utilizzare i punzoni auricolari o tag per contrassegnare individualmente 6 settimane di età C57BL / 6 topi che porto diverse microbiotas e li ospitano nella stessa gabbia. Dato che i topi sono coprofagiche e mangiare pellet fecali dal pavimento della gabbia, i microbi saranno naturalmente trasmesse orizzontalmente tra i topi. </li><li> topi Co-house per ≥1 settimana per dare il tempo per il trasferimento microbica e di cambiamento immunologico prima dell&#39;analisi. </li></ol&gt; 2. Preparazione di una cella singola sospensione dallo strato intraepiteliale piccola intestinale e lamina propria <ol><li> Preparazione delle soluzioni <ol><li> Preparare mezzi di estrazione (per tenue): 30 ml RPMI + 93 microlitri 5% (w / v) ditiotreitolo (DTT) + 60 ml di 0,5 M EDTA + 500 microlitri di siero fetale bovino (FBS). Aggiungere il DTT immediatamente prima dell&#39;uso. </li><li> Preparare supporti digestione (per intestino tenue): 25 ml RPMI + 12,5 mg dispasi + 37,5 mg di collagenasi II + 300 microlitri FBS. Aggiungere il dispasi e collagenasi immediatamente prima dell&#39;uso. </li><li> Per tutte le incubazioni eseguite a 37 ° C, le soluzioni Preriscaldate a 37 ° C. </li></ol></li><li> Eutanasia il mouse da CO 2 asfissia seguita da dislocazione cervicale. </li><li> Posizionare il lato del mouse dorsale verso il basso e spruzzare l&#39;addome con il 70% di etanolo. Utilizzare le forbici per eseguire una laparotomia dalla sequenza tagliare la pelle e la fascia poi peritoneale lungo la linea mediana ventrale from sinfisi pubica al processo xifoideo, esponendo così la cavità peritoneale. </li><li> Usare le forbici per separare il piccolo intestino dallo stomaco da sezionare lo sfintere piloro. Rimuovere delicatamente l&#39;intestino tenue dal peritoneo, prendere in giro il grasso mesenterico. <ol><li> Per rimuovere completamente l&#39;intestino tenue, effettuare un secondo taglio alla giunzione ileo-cecale. Posizionare il piccolo intestino isolato in fredda (es., 4 ° C) RPMI contenente 10% FBS per massimizzare la vitalità cellulare. </li></ol></li><li> Rimuovere delicatamente grandi pezzi di grasso con pinze curve. Usare cautela per evitare di strappare il tessuto intestinale in sé durante il tentativo di rimuovere il grasso. </li><li> Per rimuovere contenuto intestinale, intestino delicatamente a filo con 15 - 20 ml di PBS freddo utilizzando un ago di alimentazione 18 G apposto una siringa. </li><li> Usare le forbici per recidere PP, e metterli in RPMI freddo contenente il 5% di FBS. PP si trovano sul lato antimesenterico dell&#39;intestino tenue e appare come un bianco multi-lobulatimassa. A seconda del ceppo specifico, un mouse ha tipicamente 8 - 12 PP. </li><li> Tagliare il piccolo intestino in 3 - 4 segmenti pollici. </li><li> Rimuovere il grasso residuo da dolci ogni piccolo-intestinale segmento su un tovagliolo di carta inumidito con RPMI, utilizzando un bisturi noioso per prendere in giro il grasso dal tessuto. </li><li> Ruotare il tessuto all&#39;interno da cannulating segmenti intestinali con pinze curve e afferrando l&#39;estremità distale del tessuto. Quindi utilizzare un paio di pinze diritte per rimuovere delicatamente il segmento di tessuto dalle pinze curve (a partire dalla fine prossimale), con conseguente tessuto che viene invertita. </li><li> Posizionare i segmenti di tessuto in una tazza contenente 30 ml di mezzi di estrazione e un ancoretta. Fissare il coperchio sulla tazza, e mescolare a 500 rpm per 15 min a 37 ° C; agitazione dovrebbe essere vigoroso, ma non turbolenta. </li><li> Dopo l&#39;incubazione, utilizzare un filtro in acciaio per separare pezzi di tessuto dal surnatante dell&#39;epitelio contenente, che dovrebbe apparire torbida. Questo surnatante contiene ilinfociti ntraepithelial che può essere ulteriormente analizzati, se desiderato, procedendo a filtrare il campione in passi 2.18 - 2.23. </li><li> Agitare manualmente i pezzi di tessuto in RPMI per lavare via i media di estrazione residui. </li><li> Posizionare il tessuto su un tovagliolo di carta asciutto e capovolgere su se stesso più volte per facilitare la rimozione del muco residui che non è stato liberato dal mezzo di estrazione. </li><li> Posizionare frammenti di tessuto in una provetta da 1,5 ml con 600 ml di mezzo di digestione. </li><li> Usare le forbici per tritare il tessuto fino a pezzi aderiscono più alle forbici e la soluzione appare omogenea. Questo passaggio è fondamentale per garantire la completa digestione enzimatica del tessuto. </li><li> Aggiungere tenue macinata ad una tazza contenente 25 ml di mezzi di digestione. Mescolare a 500 rpm per 30 min a 37 ° C. A metà della digestione (cioè., A 15 min), pipettare su e giù con una pipetta sierologica per contribuire a spezzare eventuali grossi pezzi di tessuto. </li><li> Filtro tessuto digerito (o epithstrato Elial dal punto 2.12 se IELS di elaborazione) attraverso un colino cella di 100 micron in un tubo da 50 ml. Risciacquare il filtro con 20 ml di RPMI contenente 10% FBS. </li><li> Centrifugare la soluzione filtrata a 500 xg per 10 min a 4 ° C. </li><li> decantare con cautela il surnatante e risospendere pellet in 1 ml di RPMI contenente 10% FBS. </li><li> Filtro risospeso cellule attraverso un filtro 40 micron cellule in una provetta da 50 ml. Lavare il filtro con 20 ml di RPMI contenente 10% FBS. </li><li> Centrifugare soluzione filtrata a 500 xg per 10 min a 4 ° C. </li><li> decantare con attenzione il surnatante, e risospendere pellet in 1 ml di RPMI contenente il 2% FBS. </li></ol> 3. Preparazione di una cella sospensione singola da placche di Peyer <ol><li> Trasferimento asportato PP ad una tazza con 25 ml di mezzi di digestione e di un ancoretta. coperchio sicuro, e far girare a 500 giri al minuto per 10 minuti a 37 ° C. </li><li> Filtro digerito PP attraverso un filtro 40 micron cellule in una provetta da 50 ml. Se una qualsiasi grumi remain, premerli attraverso il filtro utilizzando l&#39;estremità piatta del pistone da una siringa da 1 ml. </li><li> Risciacquare il filtro con 10 ml di RPMI contenente 10% FBS. </li><li> Centrifugare soluzione filtrata a 500 xg per 10 min a 4 ° C. </li><li> decantare con attenzione il surnatante, e risospendere pellet in 1 ml di RPMI contenente il 2% FBS. </li></ol> 4. colorazione della superficie per citometria a flusso <ol><li> Aliquota adeguato volume delle cellule (ad es., 200 ml di LPLS) in un piatto fondo rotondo da 96 pozzetti. </li><li> Centrifugare a 500 xg per 5 minuti a 4 ° C. Decantare il surnatante invertendo la piastra. </li><li> Risospendere le cellule in 90 ml di RPMI contenente il 2% FBS e una diluizione 1: 100 di CD16 anti-/ 32 (blocco FC). Incubare per 10 minuti a 4 ° C. </li><li> Aggiungere 10 ml di PBS. Centrifugare a 500 xg per 5 minuti a 4 ° C. Decantare il surnatante invertendo la piastra. </li><li> Risospendere le cellule in 250 l di PBS. Centrifugare a 500 xg per 5 minuti a 4 ° C. surnatante decantareinvertendo la piastra. </li><li> cellule Stain (opzionale) con colorante vitalità, se lo si desidera, seguendo il protocollo del produttore. </li><li> Risospendere le cellule in 100 l 1% di formalina per fissare le cellule. Incubare per 1 ora al buio a 4 ° C. </li><li> Aggiungere 200 l di PBS. Centrifugare a 500 xg per 5 minuti a 4 ° C. Decantare il surnatante invertendo la piastra. </li><li> Risospendere le cellule in 250 l di PBS. Centrifugare a 500 xg per 5 minuti a 4 ° C. Decantare il surnatante invertendo la piastra. </li><li> Risospendere le cellule in 200 l di PBS. Analizzare le cellule utilizzando un citofluorimetro. </li></ol>

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No conflicts of interest declared.

Vi presentiamo un protocollo per l&#39;isolamento e il flusso caratterizzazione mediante citometria di piccole-intestinale linfociti, tra cui il LPLS, IELS, e linfociti nel PP. Per chi è interessato a valutare come i cambiamenti nel microbiota influenzano la piccola-intestinale sistema immunitario, ci dettaglio i semplici passaggi coinvolti nella trasmissione orizzontale di organismi tra i topi che ospitano diverse microbiotas. Sebbene questo protocollo concentra sul piccolo intestino, la procedura è la stessa per l&#...

Il compito principale del piccolo intestino è spesso considerata la digestione e l&#39;assorbimento dei nutrienti 1. Anche se questa funzione metabolica è chiaramente essenziale, il piccolo intestino ha un ruolo altrettanto significativo nel proteggere l&#39;host dallo sbarramento continuo di antigeni ambientali presenti all&#39;interno del lume 2. Il tratto intestinale separa il mondo esterno (ad es., Antigeni luminali) dall&#39;ambiente interno dell&#39;ospite con uno strato epiteliale che è soltanto un singolo strato di cellule di spessore. Come tale, il piccolo-intestinale sistema immunitario ha il compito arduo di bilanciare la soglia per la reattività, permettendo antigeni estranei dalla dieta e commensali microbi per immettere la mucosa con minima o nulla, la risposta immunitaria nel montare una risposta forte contro patogeni e altri antigeni &quot;nocivi&quot;. Risposte immunitarie eccessive o inappropriate a questi antigeni possono portare a malattie patologiche (ad es., Infiammazionimalattia Tory intestinali, diabete di tipo I, la sclerosi multipla) e deve essere evitato 3-6. Complessivamente, il tratto gastrointestinale è pensato per rappresentare il più grande organo immunitario del corpo, contenente più del 70% di tutte le cellule secernenti anticorpi 7. Il sistema immunitario del piccolo intestino è composto da 3 compartimenti principali - lamina propria (LP), lo strato intraepiteliale e placche di Peyer (PP) - che ogni contiene un particolare gruppo di linfociti 2. I linfociti LP (LPLS) sono principalmente cellule T TCRαβ con ~ cellule del 20% B; linfociti intraepiteliali (IELS) contengono pochissime cellule B con più cellule T TCRγδ di cellule T TCRαβ; e PP, che sono gli organi linfoidi secondari incorporati nel piccolo-intestinale muro, contengono cellule ~ 80% B. Anche se ciascuna di queste regioni anatomiche ha funzioni leggermente diverse e basi ontologiche, funzionano in ahmoda armonized per proteggere l&#39;host da insulti patogeni. Inoltre, vi è una crescente apprezzamento che il microbiota &#39;critico per lo sviluppo del sistema immunitario intestinale, con crescente riconoscimento del rapporto tra cognate microbi specifici e l&#39;ontogenesi di particolari linee cellulari 8,9. Inoltre, dato che l&#39;educazione del sistema immunitario intestinale colpisce risposte immunitarie nei siti anatomicamente distanti (ad es., L&#39;artrite, la sclerosi multipla, la polmonite), è diventato chiaro che lo sviluppo del sistema immunitario intestinale è rilevante per più processi di malattia rispetto al passato riconosciuto 10 -12. Come tale, l&#39;interesse per quantitativamente valutazione del sistema immunitario intestinale si è esteso al di là di interazioni ospite-patogeno per includere ora interazioni ospite-commensale e la patogenesi di molte malattie sistemiche come bene. Data la variabilità dei metodi attuali nellaisolamento dei linfociti intestinali, un metodo che è ottimizzato per la resa, la vitalità e la coerenza bilanciando il tempo richiesto è sempre più critica. I protocolli che coinvolgono gradienti di Percoll sono tempo e lavoro e potenzialmente più inclini a errori umani, che porta a rendimento variabile e la vitalità 13. Qui, forniamo un protocollo ottimizzato per l&#39;isolamento e la caratterizzazione dei linfociti da tutti e 3 i comparti del sistema immunitario del piccolo intestino. Inoltre, data crescente interesse alterazioni microbo indotte nel sistema immunitario mucosale, includiamo passi che possono essere utilizzati per consentire la trasmissione orizzontale di microrganismi tra i topi di valutare come questi cambiamenti influenzano quantitativamente il sistema immunitario intestinale.

<table><tbody><tr><td>Sterile Gloves</td><td>Kimberly-Clark</td><td>555092</td><td></td></tr><tr><td>sterile mouse cage</td><td>Innovive</td><td>MS2-AD</td><td>contains lid, cage bottom, and alpha-dri bedding</td></tr><tr><td>Metal feeder</td><td>Innovive</td><td>M-FEED</td><td></td></tr><tr><td>Water bottle</td><td>Innovive</td><td>M-WB-300</td><td></td></tr><tr><td>Card holder</td><td>Innovive</td><td>CRD-HLD-H</td><td></td></tr><tr><td>Autoclavable rodent chow (NIH-31M)</td><td>Zeigler</td><td>4131207530</td><td></td></tr><tr><td>RPMI medium 1640</td><td>Gibco</td><td>11875-119</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Sigma</td><td>D5545-5G</td><td></td></tr><tr><td>0.5 M EDTA (pH 8.0)</td><td>Ambion</td><td>AM9262</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>GemBio</td><td>100-510</td><td></td></tr><tr><td>Dispase II</td><td>Invitrogen</td><td>17105-041</td><td>the concentration in the protocol is based on an activity level of 1.878 U/mg</td></tr><tr><td>Collagenase, type II&amp;nbsp;</td><td>Invitrogen</td><td>17101-015</td><td>the concentration in the protocol is based on an activity level of 245 U/mg</td></tr><tr><td>Dissecting scissors</td><td>Roboz</td><td>RS-5882</td><td></td></tr><tr><td>Feeding needle (18 G, 2&quot; length)</td><td>Roboz</td><td>FN-7905</td><td></td></tr><tr><td>10 ml Syringe</td><td>BD</td><td>305482</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>14190-250</td><td></td></tr><tr><td>Disposable Scalpel (15 blade)</td><td>Miltex</td><td>4-415</td><td></td></tr><tr><td>Curved forceps</td><td>Roboz</td><td>RS-5211</td><td></td></tr><tr><td>Straight forceps</td><td>Roboz</td><td>RS-5132</td><td></td></tr><tr><td>Multi-purpose cups, 120 ml</td><td>VWR</td><td>89009-662</td><td></td></tr><tr><td>Stir bar</td><td>VWR</td><td>58949-062</td><td></td></tr><tr><td>Multi-position stir plate, 9-position</td><td>VWR</td><td>12621-048</td><td></td></tr><tr><td>Stainless steel conical strainer, 3 inch&amp;nbsp;</td><td>RSVP</td><td></td><td></td></tr><tr><td>1.5 ml Tube</td><td>Eppendorf</td><td>0030 125.150</td><td></td></tr><tr><td>100 &amp;mu;m Cell strainer</td><td>Falcon</td><td>08-771-19</td><td></td></tr><tr><td>40 &amp;mu;m Cell strainer</td><td>Falcon</td><td>08-771-1</td><td></td></tr><tr><td>50 ml Conical tube</td><td>Falcon</td><td>352098</td><td></td></tr><tr><td>1 ml Syringe</td><td>BD</td><td>309659</td><td></td></tr><tr><td>96-well Plate, round-bottom</td><td>Corning</td><td>3799</td><td></td></tr><tr><td>Anti-mouse CD16/32 (Fc block)</td><td>Biolegend</td><td>101320</td><td></td></tr><tr><td>(Optional) Fixable viability dye eFluor 780</td><td>eBiosciences</td><td>65-0865-18</td><td></td></tr><tr><td>10% Formalin, neutral buffered</td><td>Thermo Scientific</td><td>5725</td><td></td></tr></tbody></table>

isolation and flow cytometric characterization of murine small intestinal lymphocytes

L&#39;intestino - che contengono il maggior numero di cellule immunitarie di qualsiasi organo del corpo - sono costantemente esposti ad antigeni estranei, sia microbica e dietetici. Dato una maggiore comprensione che questi antigeni luminali contribuiscono a formare la risposta immunitaria e che l&#39;istruzione di cellule immunitarie all&#39;interno dell&#39;intestino è critica per un certo numero di malattie sistemiche, c&#39;è stato un crescente interesse nel caratterizzare il sistema immunitario intestinale. Tuttavia, molti protocolli pubblicati sono arduo e richiede tempo. Presentiamo qui un protocollo semplificato per l&#39;isolamento dei linfociti dalla propria nell&#39;intestino tenue lamina strato intraepiteliale e placche di Peyer che è rapido, riproducibile e non richiede laboriose gradienti di Percoll. Sebbene il protocollo concentra sul piccolo intestino, è adatto anche per l&#39;analisi del colon. Inoltre, si segnalano alcuni aspetti che possono avere bisogno di ulteriore ottimizzazione a seconda della specifica ques scientificozione. Questo approccio si traduce in l&#39;isolamento di un gran numero di linfociti vitali che possono poi essere utilizzati per analisi di citometria di flusso o sistemi alternativi per la caratterizzazione.

Flusso citometria di sospensioni singola cella di piccole-intestinale linfociti dovrebbe produrre una popolazione discreta di cellule che hanno caratteristiche simili in avanti e scatter laterale come splenociti (Figure 1A e 1B). I linfociti possono cominciare a morire se il tessuto non viene mantenuta a 4 ° C durante le fasi iniziali della isolamento, con conseguente popolazione linfocitaria avente un forward scatter inferiore ed essere più difficile da separare da al...

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Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

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-flow-cytometric-characterization-murine-small-intestinal

Research

JoVE Journal

Immunology and Infection

27.3K Views.  Boston Children’s Hospital. 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. 

Video: Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

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.

Isolamento e caratterizzazione di cellule dendritiche ei macrofagi dall&#39;intestino mouse

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Ricerca

Immunologia e infezioni

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Flusso Isolamento citometrica di primari murini di tipo II cellule alveolari epiteliali per gli studi funzionali e molecolari

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

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 ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

Isolando i linfociti del sistema immunitario intestinale piccolo per Mouse

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

Determinazione dei sottoinsiemi di regolamentazione delle cellule T nel timo murino, pancreatico drenante linfonodi e milza mediante citometria a flusso

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...

Flow Cytometric Characterization of Murine B Cell Development

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

Caratterizzazione citometrica a flusso dello sviluppo delle cellule B murine

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells ...

Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow cytometry. The protocol describes how to set up time mating to obtain multiple synchronous dams, the mechanical and enzymatic digestion of the pregnant uterus, the staining of single-cell suspensions, and a FACS strategy to phenotype and discriminate g1 uILCs. Although this method inevitably loses the spatial information of cellular distribution within the tissue, the protocol has been successfully applied to determine uILC heterogeneity, their response to maternal and foetal factors affecting pregnancy, their gene expression profile, and their functions.

This is a method to isolate uterine lymphoid cells from both pregnant and non-pregnant mice. This method can be used for multiple downstream applications such as FACS phenotyping, cell sorting, functional assays, RNA-seq, and proteomics. The protocol here demonstrates how to phenotype group 1 uterine innate lymphoid cells by flow cytometry.

Isolamento delle cellule linfoidi innate uterine per l'analisi mediante citometria a flusso

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow ...

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate balance of mucosal homeostasis. These 3D, multi-cellular systems offer a reductionist model for addressing multi-factorial diseases and resolving technical difficulties that arise when studying rare cell types such as tissue-resident innate lymphoid cells (ILCs). This article describes a murine system that combines small intestine organoids and small intestine lamina propria derived helper-like type-1 ILCs (ILC1s), which can be readily extended to other ILC or immune populations. ILCs are a tissue-resident population that is particularly enriched in the mucosa, where they promote homeostasis and rapidly respond to damage or infection. Organoid co-cultures with ILCs have already begun shedding light on new epithelial-immune signaling modules in the gut, revealing how different ILC subsets impact intestinal epithelial barrier integrity and regeneration. This protocol will enable further investigations into reciprocal interactions between epithelial and immune cells, which hold the potential to provide new insights into the mechanisms of mucosal homeostasis and inflammation.

This protocol offers detailed instructions for establishing murine small intestine organoids, isolating type-1 innate lymphoid cells from the murine small intestine lamina propria, and establishing 3-dimensional (3D) co-cultures between both cell types to study bi-directional interactions between intestinal epithelial cells and type-1 innate lymphoid cells.

Co-coltura di organoidi epiteliali murini dell'intestino tenue con cellule linfoidi innate

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Analisi delle sospensioni cellulari isolate da tessuti solidi da Spectral citometria a flusso

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Biologia

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, isolating intestinal lipoproteins is very difficult in vivo because they are first secreted from the small intestine into the mesenteric lymphatics. Chylomicron-containing lymph then empties into the subclavian vein from the thoracic duct to deliver components of the meal to the heart, lungs, and, ultimately, whole-body circulation. Isolating na&#239;ve chylomicrons is impossible from blood since chylomicron triglyceride undergoes hydrolysis immediately upon interaction with lipoprotein lipase and other lipoprotein receptors in circulation. Therefore, the original 2-day lymph fistula procedure, described by Bollman et al. in rats, has historically been used to isolate fresh mesenteric lymph before its entry into the thoracic vein. That protocol has been improved upon and professionalized by the laboratory of Patrick Tso for the last 45 years, allowing for the analysis of these critical lipoproteins and secretions from the gut. The Tso lymph fistula procedure has now been updated and is presented here visually for the first time. This revised procedure is a single-day surgical technique for installing a duodenal feeding tube, cannulating the mesenteric lymph duct, and collecting lymph after a meal in conscious mice. The major benefits of these new techniques include the ability to reproducibly collect lymph from mice (which harnesses the power of genetic mouse models); the reduced anesthesia time for mice during the implantation of the duodenal infusion tube and the lymph cannula; the ability to continuously sample lymph throughout the feeding and post-prandial period; the ability to quantitatively measure hormones and cytokines before their dilution and enzymatic hydrolysis in blood; and the ability to collect large quantities of lymph for isolating intestinal lipoproteins. This technique is a powerful tool for directly and quantitatively measuring dietary nutrient absorption, intestinal lipoprotein synthesis, and chylomicron secretion.

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

The present protocol describes a detailed surgical protocol for isolating flowing intestinal lymph in response to intraduodenal nutrient infusions in mice. This allows for the physiological determination of total intestinal lipid absorption and chylomicron synthesis and secretion in response to various experimental nutrients.

L'isolamento della linfa mesenterica fluente nei topi per quantificare in vivo la cinetica dell'assorbimento dei lipidi alimentari e della secrezione di chilomicroni

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, ...

Isolamento e caratterizzazione citometria a flusso di Murine Piccolo intestinale Linfociti

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Isolamento e caratterizzazione di cellule dendritiche ei macrofagi dall'intestino mouse