The authors would like to thank Dr. Sheryl Coutermarsh-Ott, Dylan McDaniel and Bettina Heid for technical discussions. The authors thank Dr. Nanda Nanthakumar for providing the Caco-2 cells. The authors also thank The Multicultural Academic Opportunities Program (MAOP) at Virginia Tech. This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Award K01DK092355 (to I.C.A.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+models+of+human+and+mouse+ductal+pancreatic+cancer.">Organoid models of human and mouse ductal pancreatic cancer.</a> Cell. 160 (1-2), 324-338 (2015).</li><li>Huch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+culture+of+genome-stable+bipotent+stem+cells+from+adult+human+liver.">Long-term culture of genome-stable bipotent stem cells from adult human liver.</a> Cell. 160 (1-2), 299-312 (2015).</li><li>Lancaster, M. 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E., Giangreco, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+the+relevance+of+human+stem+cell-derived+organoid+models+for+epithelial+translational+medicine.">Concise review: the relevance of human stem cell-derived organoid models for epithelial translational medicine.</a> Stem Cells. 31 (3), 417-422 (2013).</li><li>Kaiko, G. E., Stappenbeck, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-microbe+interactions+shaping+the+gastrointestinal+environment.">Host-microbe interactions shaping the gastrointestinal environment.</a> Trends Immunol. 35 (11), 538-548 (2014).</li><li>Kim, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitogenic+influence+of+human+R-spondin1+on+the+intestinal+epithelium.">Mitogenic influence of human R-spondin1 on the intestinal epithelium.</a> Science. 309 (5738), 1256-1259 (2005).</li><li>Chomczynski, P., Sacchi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+single-step+method+of+RNA+isolation+by+acid+guanidinium+thiocyanate-phenol-chloroform+extraction:+twenty-something+years+on.">The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on.</a> Nat Protoc. 1 (2), 581-585 (2006).</li><li>Allen, I. 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The authors declare that they have no competing financial interests.

La cultura e la manutenzione di organoidi intestinali è una procedura che può essere masterizzato da qualsiasi persona con la tecnica di coltura tissutale adeguata. Ci sono sottigliezze in passaging rispetto a crescere le cellule in un monostrato più convenzionale, ma queste sottigliezze non sono difficili da superare. I passaggi critici di questo metodo comporta poter far crescere le organoidi ad una densità sufficientemente elevata per la semina ottimale. Gli esperimenti devono essere ridimensionati con organoidi ...

La capacità di raccogliere e organoidi coltura primaria sono stati descritti per intestino tenue, colon, pancreas, fegato e cervello e sono progressi entusiasmanti germano per comprendere un fenomeno fisiologicamente più rappresentativo per la biologia dei tessuti 1-5. I primi metodi che descrivono la cultura e la manutenzione di piccoli organoidi intestinale è stata riportata da Sato et al. Fuori dal laboratorio di Hans Clevers 1. Prima di questo metodo, la raccolta e la coltura di cellule epiteliali intestinali primarie ha dimostrato di essere limitato e inefficace nel sostenere la crescita delle cellule epiteliali. Metodi incluse dissociazione del tessuto tramite incubazione con enzimi, come collagenasi e dispasi, che finirebbe per portare alla conseguenza di fibroblasti primari mescolati 6. Queste condizioni potrebbero anche essere il momento limitati a sostenere la coltura delle cellule epiteliali. Minimo a nessun nicchia delle cellule epiteliali possano costituire, come le cellule epiteliali sarebbero entrati a causa di apoptosila mancanza di fattori di crescita appropriati o perdita di integrità contatto, definito anokis 7. L&#39;avvento del sistema di coltura 3D-organoide ha fornito un metodo per cellule intestinali primarie coltura contenenti una gamma di tipi di cellule intestinali in coltura sostenuta 1. Questi organoidi epiteliali presentano vantaggi rispetto alle linee cellulari è che essi sono composti da diverse cellule differenziate, e meglio imitano l&#39;organo che sono derivati ​​da 8 in vivo. Il processo di definitiva &quot;crescere un mini budello in un piatto&quot; ha dimostrato di essere un valido strumento per valutare la risposta di epitelio intestinale sotto diversi stimoli. Indagare l&#39;interazione delle cellule intestinali primarie con modelli patogeni microbici associati molecolari (PAMPs) è rilevante per il campo dell&#39;immunologia come questi modelli molecolari in grado di regolare diverse risposte sia da host e microbo 9. Non solo è possibile investigatori ora esplorare queste interazioni con organoidi del mouse, ma hannopuò essere colta da esseri umani come ben 2. Questa tecnologia ha il potenziale per alterare radicalmente la medicina personalizzata e si è tentati di speculare sui progressi che questa tecnica renderà possibile in un prossimo futuro. L&#39;obiettivo generale di questo metodo è quello di fornire un protocollo per la coltura, l&#39;espansione e il trattamento di organoidi intestinali con una varietà di stimoli. Tali stimoli possono infine variare da vaccini, PAMPs batteriche, agenti patogeni dal vivo, gastrointestinale (GI) e cancro terapeutica. L&#39;isolamento e la coltura di topo organoidi intestinali è stato adattato da Sato et al. Anche se ci sono lievi deviazioni dal metodo originale, il prodotto finale è organoide cultura è ancora raggiunto quando si segue questo protocollo. Questo metodo è focalizzato sulla descrizione una tecnica adeguata per una corretta normalizzazione quando si lavora con strutture cellulari non omogenei, che devono essere presi in considerazione quando condurre un saggio basato su cellule nterra d&#39;ombra.

<table>
	<tbody>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11050</td>
			<td>(Section 1,3,6) Or equivalent brand</td>
		</tr>
		<tr>
			<td>Sorvall Legend XTR Centrifuge</td>
			<td>Thermo</td>
			<td></td>
			<td>(Section 1,3)</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>GE Healthcare</td>
			<td>Sh30243.01</td>
			<td>(Section 1,6) For Caco-2 and HEK293 Rspondin1 cells</td>
		</tr>
		<tr>
			<td>HEK293T-Rspondin1 secreting cell line</td>
			<td></td>
			<td></td>
			<td>(Section 1) Described and modified from Kim, K.A. et al. Lentiviral particles contained RSPO1(NM_138683) ORF cDNA cloned into a pReceiver-Lv105 backbone custom ordered and purchased from GeneCopoeia.&#160;</td>
		</tr>
		<tr>
			<td>50 ml conical tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td>(Section 1) Or equivalent brand</td>
		</tr>
		<tr>
			<td>T-175 Flask</td>
			<td>Corning</td>
			<td>431079</td>
			<td>(Section 1) Or equivalent brand&#160;</td>
		</tr>
		<tr>
			<td>Protein Matrix</td>
			<td>Corning</td>
			<td>356231</td>
			<td>(Section 2,3,4,5,6) Matrigel Growth Factor Reduced&#160;</td>
		</tr>
		<tr>
			<td>HyClone Dulbecco&#39;s (DPBS)</td>
			<td>GE Healthcare</td>
			<td>SH30264.01</td>
			<td>(Section 2,3)</td>
		</tr>
		<tr>
			<td>DMEM/F12&#160;</td>
			<td>Life Technologies</td>
			<td>12634-010</td>
			<td>(Section 2,3) Advanced DMEM/F12</td>
		</tr>
		<tr>
			<td>Corning 24 Well TC Plates</td>
			<td>Corning</td>
			<td>3524</td>
			<td>(Section 2)</td>
		</tr>
		<tr>
			<td>N2 Supplement 100x&#160;</td>
			<td>Life Technologies</td>
			<td>17502-048</td>
			<td>(Section 2)</td>
		</tr>
		<tr>
			<td>B27 without vitamin A 50x&#160;</td>
			<td>Life Technologies</td>
			<td>12587-010&#160;</td>
			<td>(Section 2)</td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Life Technologies</td>
			<td>15596-026</td>
			<td>(Section 2)</td>
		</tr>
		<tr>
			<td>Glutamine Supplement (Glutamax)</td>
			<td>Life Technologies</td>
			<td>35050-061</td>
			<td>(Section 2) Can Combine with Advanced DMEM/ F12</td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Life Technologies</td>
			<td>15630-080</td>
			<td>(Section 2) Can Combine with Advanced DMEM/ F12&#160;</td>
		</tr>
		<tr>
			<td>10ml Serological Pipet</td>
			<td>Falcon</td>
			<td>357551</td>
			<td>(Section 2) Or equivalent brand</td>
		</tr>
		<tr>
			<td>Murine Noggin</td>
			<td>Peprotech</td>
			<td>250-38</td>
			<td>(Section 2) Stock = 100 mg/ml</td>
		</tr>
		<tr>
			<td>N-Acetyl-L-cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165</td>
			<td>(Section 2) Stock = 1M</td>
		</tr>
		<tr>
			<td>Recombinant Mouse EGF</td>
			<td>Biolegend</td>
			<td>585608</td>
			<td>(Section 2) Stock = 500 mg/ml</td>
		</tr>
		<tr>
			<td>Rocker Variable</td>
			<td>Bioexpres</td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>dissecting scissors</td>
			<td></td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>forceps</td>
			<td></td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>glass slides</td>
			<td></td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>dissecting tweezers</td>
			<td></td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>25 ml Serological Pipet</td>
			<td>Falcon</td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>EDTA&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>SLBB9821</td>
			<td>(Section 3) 0.5M or alternative TC grade EDTA</td>
		</tr>
		<tr>
			<td>Sterile Petri Dish 100mm x 15mm</td>
			<td>Fisher</td>
			<td>FB0875712</td>
			<td>(Section 3) Or equal sized TC dish</td>
		</tr>
		<tr>
			<td>1ml Syringe</td>
			<td>Becton Dickinson</td>
			<td>309659</td>
			<td>(Section 4)</td>
		</tr>
		<tr>
			<td>Precision Glide Needle</td>
			<td>Becton Dickinson</td>
			<td>305120</td>
			<td>(Section 4) 23G x 1 1/4 (0.6mm x 30mm)</td>
		</tr>
		<tr>
			<td>Flagellin from Bacillus subtilis</td>
			<td>Invivogen</td>
			<td>tlrl-bsfla&#160;</td>
			<td>(Section 5,6)</td>
		</tr>
		<tr>
			<td>Listeria monocytogenes</td>
			<td>ATCC</td>
			<td>19115</td>
			<td>(Section 5,6)&#160;(Murray&#160;et&#160;al.)&#160;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629-1EA</td>
			<td>(Section 5,6)Or equivalent brand</td>
		</tr>
		<tr>
			<td>BBL Brain Heart Infusion Agar</td>
			<td>Becton Dickinson</td>
			<td>211065</td>
			<td>(Section 5)</td>
		</tr>
		<tr>
			<td>Bacto Brain Heart Infusion</td>
			<td>Becton Dickinson</td>
			<td>237500</td>
			<td>(Section 5)</td>
		</tr>
		<tr>
			<td>Caco-2</td>
			<td>ATCC</td>
			<td>HTB-37</td>
			<td>(Section 6)</td>
		</tr>
		<tr>
			<td>Trypsin&#160;</td>
			<td>gibco</td>
			<td>25200056</td>
			<td>(section 6)</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>A412-4</td>
			<td>(Section 6)</td>
		</tr>
		<tr>
			<td>SpectraMax M5</td>
			<td>Molecuar Devices</td>
			<td></td>
			<td>(Section 6)</td>
		</tr>
		<tr>
			<td>96 Well Assay Plate</td>
			<td>Corning</td>
			<td>3603</td>
			<td>(Section 6) Black Plate, Clear Bottom TC treated</td>
		</tr>
		<tr>
			<td>Nuclear Staining Dye</td>
			<td>Life Technologies</td>
			<td>H1399</td>
			<td>(section 6) Hoechst 33342</td>
		</tr>
		<tr>
			<td>T-75 Flask</td>
			<td>Corning</td>
			<td>430641</td>
			<td>(Section 6) Or equivalent brand</td>
		</tr>
		<tr>
			<td>15 ml conical tube</td>
			<td>Falcon</td>
			<td>352096</td>
			<td>(Section1,3) Or equivalent brand</td>
		</tr>
		<tr>
			<td>1.7 ml polypropylene tube</td>
			<td>Bioexpress</td>
			<td>C-3262-1</td>
			<td>Or equivalent brand</td>
		</tr>
		<tr>
			<td>Quick-RNA MiniPrep</td>
			<td>Zymo Research</td>
			<td>R1054</td>
			<td>Or equivalent brand</td>
		</tr>
		<tr>
			<td>TNF-alpha&#160;</td>
			<td>Applied Biosystems</td>
			<td>Mm 00443260_g1</td>
			<td>Taqman gene expression assay kit</td>
		</tr>
		<tr>
			<td>IL-6&#160;</td>
			<td>Applied Biosystems</td>
			<td>(Mm 00446190_m1</td>
			<td>Taqman gene expression assay kit</td>
		</tr>
		<tr>
			<td>IL-1beta</td>
			<td>Applied Biosystems</td>
			<td>Mm 00434228_m1</td>
			<td>Taqman gene expression assay kit</td>
		</tr>
		<tr>
			<td>IL-18</td>
			<td>Applied Biosystems</td>
			<td>Mm 00434225_m1</td>
			<td>Taqman gene expression assay kit</td>
		</tr>
		<tr>
			<td>18s</td>
			<td>Applied Biosystems</td>
			<td>Hs 99999901_s1</td>
			<td>Taqman gene expression assay kit</td>
		</tr>
		<tr>
			<td>7500 Fast Real Time PCR System</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nexus gradient Mastercycler</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Fast Universal PCR Master Mix</td>
			<td>Life Technologies</td>
			<td>4352042</td>
			<td></td>
		</tr>
		<tr>
			<td>High Capacity cDNA Reverse Transcription Kit</td>
			<td>Life Technologies/Applied Biosystems</td>
			<td>4368814</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Optical 96-Well Reaction Plate, 0.1 mL</td>
			<td>Life Technologies/Applied Biosystems</td>
			<td>4346907</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse R-Spondin 1 Protein</td>
			<td>R&#38;D Systems</td>
			<td>3474-RS-050</td>
			<td>500 ng/ml</td>
		</tr>
		<tr>
			<td>chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>C7559</td>
			<td></td>
		</tr>
	</tbody>
</table>

the ex vivo culture and pattern recognition receptor stimulation of mouse intestinal organoids

organoidi intestinali primarie sono un modello di sistema di valore che ha il potenziale per avere un impatto significativo nel campo delle mucose immunologia. Tuttavia, la complessità delle caratteristiche di crescita organoide portano avvertimenti significativi per l&#39;investigatore. In particolare, i modelli di crescita di ogni singolo organoide sono molto variabili e creano una popolazione eterogenea di cellule epiteliali in coltura. Con tali limitazioni pratiche coltura tissutale comune non possono essere semplicemente applicate al sistema organoide a causa della complessità della struttura cellulare. Conteggio e placcatura basato esclusivamente sul numero di cellule, che è comune a cellule separate individualmente, quali le linee cellulari, non è un metodo affidabile per organoidi se viene applicata una tecnica di normalizzazione. Normalizzazione al contenuto totale di proteine ​​è fatta complessa a causa della matrice proteica residente. Queste caratteristiche in termini di numero di cellule, forma e tipo di cellula dovrebbero essere presi in considerazione quando si valuta con secretotende dalla massa organoide. Questo protocollo è stato generato per delineare una procedura semplice per la cultura e il trattamento di piccoli organoidi intestinale con agenti patogeni microbici e pamp (PAMPs). Si sottolinea, inoltre, le tecniche di normalizzazione che dovrebbero essere applicate quando l&#39;analisi delle proteine ​​sono condotte dopo una tale sfida.

Quando si segue questo protocollo per coltivare organoidi intestinali, caratteristica a forma di sfera organoidi saranno presenti dopo la raccolta. L&#39;aggiunta di R-spondin1 mezzi condizionati ogni giorno avvierà la crescita e la gemmazione delle organoidi. La crescita di organoidi è mostrato in Figura 1A - F e rappresentativa organoidi intestinali nei giorni 1, 2, 4, 5, 6 e il giorno 14. Figura 1F rappresenta le ca...

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The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Qui, un protocollo per raccogliere, gestire e trattare il mouse piccoli organoidi intestinale con modelli patogeni associati molecolari (PAMPs) e Listeria monocytogenes è descritto, così come l&#39;accento sulla espressione genica e corrette tecniche di normalizzazione per le proteine.

Il<em&gt; Ex Vivo</em&gt; Cultura e Pattern Recognition recettore stimolazione del mouse intestinale organoidi

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

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Research

JoVE Journal

Immunology and Infection

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

13.5K Views.  Virginia Maryland College of Veterinary Medicine, Virginia Tech.  Qui, un protocollo per raccogliere, gestire e trattare il mouse piccoli organoidi intestinale con modelli patogeni associati molecolari (PAMPs) e Listeria monocytogenes è descritto, così come l'accento sulla espressione genica e corrette tecniche di normalizzazione per le proteine. 

Video: The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

Valutare l&#39;innato rilevamento dell&#39;infezione da HIV-1 CD4 infetti<sup&gt; +</sup&gt; Cellule T dalle cellule dendritiche plasmacitoidi Utilizzando un<em&gt; Ex vivo</em&gt; Sistema di co-coltura.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Ricerca

Immunologia e infezioni

Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, the PRRs RIG-I and PKR bind to specific viral RNA ligands. This first mediates conformational switching and oligomerization, and then enables activation of an antiviral interferon response. While methods to measure antiviral host gene expression are well established, methods to directly monitor the activation states of RIG-I and PKR are only partially and less well established.
Here, we describe two methods to monitor RIG-I and PKR stimulation upon infection with an established interferon inducer, the Rift Valley fever virus mutant clone 13 (Cl 13). Limited trypsin digestion allows to analyze alterations in protease sensitivity, indicating conformational changes of the PRRs. Trypsin digestion of lysates from mock infected cells results in a rapid degradation of RIG-I and PKR, whereas Cl 13 infection leads to the emergence of a protease-resistant RIG-I fragment. Also PKR shows a virus-induced partial resistance to trypsin digestion, which coincides with its hallmark phosphorylation at Thr 446. The formation of RIG-I and PKR oligomers was validated by native polyacrylamide gel electrophoresis (PAGE). Upon infection, there is a strong accumulation of RIG-I and PKR oligomeric complexes, whereas these proteins remained as monomers in mock infected samples.
Limited protease digestion and native PAGE, both coupled to western blot analysis, allow a sensitive and direct measurement of two diverse steps of RIG-I and PKR activation. These techniques are relatively easy and quick to perform and do not require expensive equipment.

Innate defenses to virus infections are triggered by pattern recognition receptors (PRRs). The two cytoplasmic PRRs RIG-I and PKR bind to viral signature RNAs, change conformation, oligomerize, and activate antiviral signaling. Methods are described which allow to conveniently monitor the conformational switching and the oligomerization of these cytoplasmic PRRs.

Attivazione Monitoraggio del Pattern Recognition antivirale recettori RIG-I E PKR By limitata proteasi Digestione e PAGE Native

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, ...

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

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

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

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

Linfociti Isolamento da Pelle umana per l&#39;analisi fenotipica e<em&gt; Ex Vivo</em&gt; Cell Culture

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

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

Organoidi come modello per le malattie infettive: cultura dei diritti dell&#39;uomo e organoidi stomaco murini e microiniezione dell&#39;Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

Isolamento e<em&gt; Ex Vivo</em&gt; Cultura di Vδ1<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt; Celle γδ T, un Extrathymic αβT-cellule progenitrici

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between the T-cell receptor and tumor antigens, presented by human leukocyte antigens, governs T-cell functionality; these methods include protein production, X-ray crystallography, biophysics, and functional T-cell experiments.

Utilizzando cristallografia a raggi X, Biofisica, e saggi funzionali per determinare i meccanismi di amministrazione delle cellule T del recettore Riconoscimento di cancro antigeni

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell ...

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

The intestine displays an architecture of repetitive crypt structures consisting of different types of epithelial cells, lamina propia containing immune cells, and stroma. All of these heterogeneous cells contribute to intestinal homeostasis and participate in antimicrobial host defense. Therefore, identifying a surrogate model for studying immune response and antimicrobial activity of the intestine in an in vitro setting is extremely challenging. In vitro studies using immortalized intestinal epithelial cell lines or even primary crypt organoid culture do not represent the exact physiology of normal intestine and its microenvironment. Here, we discuss a method of culturing mouse colon tissue in a culture dish and how this ex vivo organ culture system can be implemented in studies related to antimicrobial host defense responses. In representative experiments, we showed that colons in organ culture express antimicrobial peptides in response to exogenous IL-1&#946; and IL-18. Further, the antimicrobial effector molecules produced by the colon tissues in the organ culture efficiently kill Escherichia coli in vitro. This approach, therefore, can be utilized to dissect the role of pathogen- and danger-associated molecular patterns and their cellular receptors in regulating intestinal innate immune responses and antimicrobial host defense responses.

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

The ex vivo organ culture allows investigation of biological processes in the context of the intact tissue architecture. Here, we introduce a method of ex vivo culture of the mouse colon, which can be used to study innate immunity and antimicrobial host defense in the intestine.

Il<em&gt; Ex Vivo</em&gt; Colon Organ Culture e il suo uso in Antimicrobial Host Defense Studies

The intestine displays an architecture of repetitive crypt structures consisting of different types of epithelial cells, lamina propia containing immune ...

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 ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Un modello di cultura Bursal-cellula primaria di pollo Ex Vivo per studiare la patogenesi Virus bursite infettiva

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Formazione immagine confocal di RNA Double-Stranded e Pattern Recognition Receptors in infezione Virus RNA negativo

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

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Valutare l'innato rilevamento dell'infezione da HIV-1 CD4 infetti

Attivazione Monitoraggio del Pattern Recognition antivirale recettori RIG-I E PKR By limitata proteasi Digestione e PAGE Native

Linfociti Isolamento da Pelle umana per l'analisi fenotipica e

Organoidi come modello per le malattie infettive: cultura dei diritti dell'uomo e organoidi stomaco murini e microiniezione dell'Helicobacter Pylori

Isolamento e

Utilizzando cristallografia a raggi X, Biofisica, e saggi funzionali per determinare i meccanismi di amministrazione delle cellule T del recettore Riconoscimento di cancro antigeni

Il

Isolando i linfociti del sistema immunitario intestinale piccolo per Mouse

Un modello di cultura Bursal-cellula primaria di pollo Ex Vivo per studiare la patogenesi Virus bursite infettiva

Formazione immagine confocal di RNA Double-Stranded e Pattern Recognition Receptors in infezione Virus RNA negativo