Gli autori desiderano ringraziare di Sally Roberts (University of Birmingham, Birmingham, UK) per la gentile dono dell&#39;anticorpo E1E4. Questo lavoro è stato sostenuto da una borsa di studio dal National Cancer Institute (4R00CA137160-03).

1. Preparazione Culture Raft organotipiche <ol><li> Le griglie metalliche zattera molto prima essere trattata con acido cromico solforico per rimuovere eventuali residui che potrebbero interferire con il processo di differenziazione. Griglie metalliche immergere in un bicchiere di vetro contenente acido solforico per un&#39;ora, quindi risciacquati in continuo per tutta la notte con acqua di rubinetto. Dopo il risciacquo durante la notte, griglie zattera devono essere risciacquati per 3-5 ore in acqua bidistillata. </li><li> Per fornire il supporto per le zattere, piegare tre lati della zattera circa 0,5 cm a uguale distanza l&#39;uno dall&#39;altro. Le griglie di metallo dovrebbe essere sterilizzati in autoclave. </li><li> Preparare il tampone 10X ricostituzione aggiungendo 2,2 g di NaHCO3 e 4,8 g Hepes. Sciogliere in 100 ml di 0,05 M NaOH. Filtrare sterilizzare e conservare in aliquote di 5 ml a -20 ° C. </li><li> Preparare il 10X DMEM senza bicarbonato di sodio. Filtrare sterilizzare e conservare in aliquote di 5 ml a -20 ° C. </li><li> Preparare il fattore di crescita epidermico mouse: Sciogliere 100 mg di EGF e 10 mg di BSA ciascuna in 10 ml ddh 2 0 deionizzata. Combinare EGF e BSA e aggiungere 80 ml ​​di acqua deionizzata ddh 2 0 per un volume totale di 100 ml. Filtro-sterilizzare, aliquota (5 ml) e conservare in -20 ° C. Per ogni litro di mezzo E aggiungono 5 ml di 1 mg / ml di EGF (concentrazione finale 5 ng / ml). </li></ol> 2. Preparazione del gel di collagene <ol><li> Un gel di collagene è richiesto per ogni zattera. Il collagene deve essere tenuti in ghiaccio per evitare che la solidificazione. Ogni gel di collagene prevede 3 ml di miscela consistente in collagene: 2,4 ml di freddo Rat-tail collagene di tipo I (concentrazione finale di 3-4 mg / ml), 0,3 ml di tampone di ricostituzione 10X, 10X 0,3 ml DMEM e1-2 x 10 6 topo J2 fibroblasti 3T3 come cellule di alimentazione. Per le soluzioni ad alta concentrazione di collagene ratto coda, diluire in 0,02 M di acido acetico per ottenere la corretta concentrazione di lavoro. </li><li> Determinare il numero di gel di collagene (zattere) avrete bisogno e quindi calcolare una master mix per la quantità di collagene, reconstit 10Xtampone distribuzione pesi, 10X DMEM e J2 fibroblasti richiesto. Include sufficiente in ciascun master mix per 3-4 gel extra a causa della viscosità del collagene. </li><li> Per preparare il gel di collagene, Tripsinizzare le J2 fibroblasti e neutralizzare con medium. Mescolare tutti i fibroblasti e metterlo in un flaconcino da 50 ml. Contare le cellule per determinare il numero delle zattere che possono essere apportate. Ancora, 1-2x10 6 fibroblasti sono necessari per gel di collagene e quindi per zattera. Centrifugare le cellule a bassa velocità. </li><li> Aspirare il mezzo dal fibroblasti pellet e risospendere in quantità appropriata di tampone di ricostituzione 10X, 10X DMEM e collagene per il numero delle zattere calcolati. Aggiungere il collagene ultimo per evitare che il gel di solidificazione troppo rapidamente. Una volta che il collagene è aggiunto, mescolare velocemente, ma con cura per evitare l&#39;introduzione di bolle nel gel. Il gel deve essere un colore rosso arancio, indicativo del pH corretto. Se la miscela di gel è troppo gialla, aggiungere un paio di gocce di filtro sterilizzato 1NNaOH e mescolare delicatamente per ottenere il colore corretto. </li><li> Aggiungere 3 ml di collagene: miscela fibroblasti nei pozzetti di una piastra da coltura da 6 pozzetti. La miscela di collagene devono essere pipettati lentamente lungo il lato di ogni bene per evitare di generare bolle. Porre la piastra a 37 ° C tessuto incubatore per colture e far solidificare per 30 minuti. </li><li> Dopo 30 minuti, aggiungere 3 ml di terreno E con EGF per la parte superiore del gel di collagene solidificato e riportare la piastra nell&#39;incubatore. I gel devono essere mantenute a 37 ° C per almeno un giorno, ma entro quattro giorni. I risultati ottimali si verificano dopo due giorni di incubazione. </li></ol> 3. Cheratinociti Preparazione per la differenziazione <ol><li> Cheratinociti passaggio bassi dovrebbe essere cresciuto fino a circa il 70% di confluenza. Ogni zattera richiede 1-2x10 6 cheratinociti. Tripsinizzare dei cheratinociti e neutralizzare con media E con EGF. Contare le cellule per determinare il numero delle zattere che possono essere apportate. Centrifugare le cellule a bassa speed. Risospendere il pellet in 1 ml di terreno E con EGF per ogni coltura zattera calcolato. </li><li> Aspirare il mezzo dai gel di collagene inclinando la piastra da 6 pozzetti. Aggiungere 2 ml di terreno fresco con EGF E ad ogni gel. Per ogni gel, accuratamente aggiungere 1 ml di cheratinociti risospeso a 2 ml di terreno E già presenti nel pozzo. Pipettare delicatamente i cheratinociti più il 2 ml di media e poi lasciato cadere goccia a goccia di nuovo sul gel. Agitando o oscillare la piastra può provocare distribuzione irregolare delle cellule a un lato del gel di collagene. Collocare la piastra nel 37 ° C incubatore. </li><li> Le cellule devono essere coltivate a confluenza, sostituendo il mezzo E giornaliera. Le cellule sono confluenti quando le modifiche medio giallo per un giorno dopo un cambiamento in mezzo. Questo richiede di solito 2-4 giorni. Se il mezzo non è cambiato da quattro giorni, procedere al passo successivo. </li></ol> 4. Esecuzione delle Culture Raft <ol><li> Utilizzare pinze sterili per inserire un sterile griglia zattera di metallo, piegato verso il basso i lati, In un piatto 10 cm. Aspirare il mezzo di gel di collagene. Per allentare il gel dalle pareti del pozzo, passare attorno al perimetro del gel con una spatola sterile utilizzando un movimento su e giù. Per rimuovere il gel di collagene, inclinare leggermente la piastra e sollevare mettendo sotto una spatola. Disporre il gel di collagene sulla griglia metallica senza generare bolle tra la griglia e il gel. Due gel può essere posizionato su ogni griglia. </li><li> Al fine di effettuare una interfaccia aria-liquido, aggiungere mezzo senza EGF E lentamente verso il fondo del piatto in modo che la griglia zattera sta toccando i supporti, ma il gel di collagene non è. I media non devono entrare attraverso i fori nella griglia. Aggiungere lentamente il mezzo per evitare di generare bolle sotto la griglia, in modo da evitare differenziazione uniforme. Incubare le zattere a 37 ° C e modificare il mezzo ogni altro giorno mantenendo l&#39;interfaccia aria-liquido. Raccogliere le zattere dopo 14 giorni. </li></ol> 5. Risultati rappresentativi Quando tegli protocollo viene eseguito correttamente con cheratinociti primari, questa procedura si tradurrà in un ben differenziato epitelio in cui i diversi strati della pelle sono evidenti. Questo essere ottenuto attraverso l&#39;esame istologico di colorazione sezioni di colture zattera ematossilina ed eosina (EE), come mostrato in figura 1B. Colorazione H &amp; E è anche utile esaminare la morfologia zattere e l&#39;effetto di virus o composti antivirali sulla capacità delle cellule di stratificare e differenziare. Ad esempio, zattere fatte da HPV-immortalizzate cheratinociti umani primari (HFK-31) sono notevolmente più spesso rispetto alle zattere fatte dal prepuzio cheratinociti umani normali (HFK), riflettendo un aumento del tasso di proliferazione e la capacità delle proteine ​​di HPV per mantenere le cellule differenzianti attivo nel ciclo cellulare (figura 1B) 10. Ciò si traduce in ritenzione nuclei tutta dell&#39;epitelio, mentre cheratinociti normali uscire dal ciclo cellulare sulla differenziazione, con conseguente rottura delinvolucro nucleare. Per approfondire la differenziazione, l&#39;immunoistochimica può essere eseguito per esaminare marcatori di differenziazione specifiche, incluse citocheratine, così come involucrina, e filaggrina. Citocheratine Diverse sono espressi in specifici momenti di differenziazione 7. Come mostrato in figura 2, citocheratina 10 (K10) non è espressa nello strato basale, e si trova solo negli strati soprabasali che consistono di cellule progressivamente differenziazione. In zattere HPV positivi, l&#39;espressione di K10 è ritardato rispetto a quella dei cheratinociti normali, nuovamente riflettono l&#39;influenza di proteine ​​HPV sulla differenziazione dei cheratinociti. Immunoistochimica può anche essere usato per esaminare il profilo di espressione di geni virali. In figura 3 è una colorazione rappresentativo esaminare l&#39;espressione della proteina HPV E1 ^ E4, che è una proteina in ritardo la cui espressione è limitata alle strati superiori dell&#39;epitelio. Come previsto, non si colorazione osservata in colture zattera dal normale human cheratinociti prepuzio. <img alt="Figura 1" src="/files/ftp_upload/3668/3668fig1.jpg" /> Figura 1. Schema A) del procedimento per la preparazione di colture di cheratinociti zattera organotipiche primarie o linee cellulari epiteliali. B) colorazione ematossilina ed eosina delle culture zattera organotipiche generati da cheratinociti del prepuzio umano mantenendo stabilmente HPV-31 (HFK-31) episomi, o da cheratinociti umani (HFK prepuzio). I singoli strati epiteliali sono identificati, nonché la spina collagene. <img alt="Figura 2" src="/files/ftp_upload/3668/3668fig2.jpg" /> Figura 2. Cytokeratin 10 (K10) espressione è limitata agli strati soprabasali dell&#39;epitelio. L&#39;immunoistochimica è stata eseguita su sezioni di culture zattera organotipiche generati da HFK-31 cellule, nonché HFKs normali, utilizzando un anticorpo a K10. Il DNA cellulare è stato controcolorate con DAPI. Le immagini sono state catturate utilizzando confLOCALE microscopia a fluorescenza. Le frecce indicano lo strato basale dell&#39;epitelio. <img alt="Figura 3" src="/files/ftp_upload/3668/3668fig3.jpg" /> Figura 3. La proteina E1 HPV ^ E4 è prodotta ritardo nella fase produttiva del ciclo vitale del virus. L&#39;immunoistochimica è stata eseguita su sezioni di culture zattera organotipiche generati da HFK-31 cellule, nonché HFKs normali usando anticorpi E1E4. Il DNA cellulare è stato controcolorate con DAPI. Le immagini sono state catturate utilizzando la microscopia confocale a fluorescenza. Le frecce indicano lo strato basale dell&#39;epitelio.

<ol>
	<li>Andrei, G., Duraffour, S., VandenOord, J., Snoeck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+raft+cultures+for+investigations+of+virus+growth,+pathogenesis+and+efficacy+of+antiviral+agents.">Epithelial raft cultures for investigations of virus growth, pathogenesis and efficacy of antiviral agents.</a> Antiviral Research. 85, 431-449 (2010).</li><li>Asselineau, D., Prunieras, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+'simplified'+skin:+control+of+fabrication.">Reconstruction of 'simplified' skin: control of fabrication.</a> The British Journal of Dermatology. 111, 219-222 (1984).</li><li>Bodily, J., Alam, S., Chen, H., Meyers, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organotypic epithelial raft cultures and the study of the natural history of human papillomavirus. , (2006).</li><li>Cheng, S., Schmidt-Grimminger, D. C., Murant, T., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation-dependent+up-regulation+of+the+human+papillomavirus+E7+gene+reactivates+cellular+DNA+replication+in+suprabasal+differentiated+keratinocytes.">Differentiation-dependent up-regulation of the human papillomavirus E7 gene reactivates cellular DNA replication in suprabasal differentiated keratinocytes.</a> Genes &#38; Development. 9, 2335-2349 (1995).</li><li>Dollard, S. C., Wilson, J. L., Demeter, L. M., Bonnez, W. R., Reichman, C., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+human+papillomavirus+and+modulation+of+the+infectious+program+in+epithelial+raft+cultures.+OFF.">Production of human papillomavirus and modulation of the infectious program in epithelial raft cultures. OFF.</a> Genes &#38; Development. 6, 1131-1142 (1992).</li><li>Frattini, M. G., Lim, H. B., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+synthesis+of+oncogenic+human+papillomaviruses+requires+episomal+genomes+for+differentiation-dependent+late+expression.">In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation-dependent late expression.</a> Proceedings of the National Academy of Sciences of the United States of America. 93, 3062-3067 (1996).</li><li>Kopan, R., Traska, G., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoids+as+important+regulators+of+terminal+differentiation:+examining+keratin+expression+in+individual+epidermal+cells+at+various+stages+of+keratinization.">Retinoids as important regulators of terminal differentiation: examining keratin expression in individual epidermal cells at various stages of keratinization.</a> The Journal of Cell Biology. 105, 427-440 (1997).</li><li>Longworth, M. S., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+human+papillomaviruses+in+differentiating+epithelia.">Pathogenesis of human papillomaviruses in differentiating epithelia.</a> Microbiology and Molecular Biology Review. 68, 362-372 (2004).</li><li>Meyers, C., Frattini, M. G., Hudson, J. B., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthesis+of+human+papillomavirus+from+a+continuous+cell+line+upon+epithelial+differentiation.">Biosynthesis of human papillomavirus from a continuous cell line upon epithelial differentiation.</a> Science. 257, 971-973 (1992).</li><li>Wilson, R., Laimins, L. 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> Differentiation of HPV-Containing Cells Using Organotypic Raft Culture or Methylcellulose. , 119-119 (2006).</li><li>zur Hausen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Papillomavirus+infections--a+major+cause+of+human+cancers.">Papillomavirus infections--a major cause of human cancers.</a> Biochimica et Biophysica Acta. 1288, F55-F78 (1996).</li></ol>

Non abbiamo nulla da rivelare.

Descriviamo qui un metodo che può essere usato per studiare differenziazione epiteliale in generale, ma può anche essere facilmente adattato per studiare la patogenesi virale, come pure l&#39;efficacia della terapia potenziali. Molti virus bersaglio le cellule epiteliali sia come sito primario di infezione, come nel caso di HPV, o ad un certo punto nel ciclo vitale del virus, come con herpesvirus. Sebbene in crescita culture zattera organotipiche richiede molto tempo, la capacità di ricapitolare fedelmente in viv...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Rat Tail collagene di tipo 1 (4-5mg/ml) </td><td> BD Biosciences </td><td> 354236 </td></tr><tr><td> DMEM senza NaHC0 3 </td><td> Invitrogen </td><td> 12100-061 </td></tr><tr><td> NaHC0 3 </td><td> Sigma </td><td> S5761 </td></tr><tr><td> Hepes </td><td> Calbiochem </td><td> 391338 </td></tr><tr><td> Griglie metalliche in acciaio inox </td><td> Williams e Mettle Co. </td><td> CR-03063-040-100-S </td></tr><tr><td> E medium 11 </td><td></td><td></td></tr><tr><td> Epidermal Growth Factor </td><td> BD Biosciences </td><td> 354010 </td></tr></tbody></table>

generation of organotypic raft cultures from primary human keratinocytes

Lo sviluppo di culture organotipiche zattera epiteliali ha fornito ai ricercatori un efficiente sistema in vitro, che riassume fedelmente la differenziazione epiteliale. Ci sono molti usi per questo sistema. Ad esempio, la capacità di crescere colture tridimensionali zattera organotipiche di cheratinociti è un passo importante nello studio di papillomavirus umano (HPV) 1. Il ciclo di vita di HPV è strettamente legata alla differenziazione dell&#39;epitelio squamoso 2. Organotipiche culture zattera epiteliali, come dimostrato qui riprodurre l&#39;intero ciclo di vita papillomavirus, compresa la produzione di virus 3,4,5. In aggiunta, queste culture zattera presentano lesioni displastiche simili a quelli osservati al momento in vivo con infezione da HPV. Quindi questo sistema può anche essere usato per studiare il cancro delle cellule epiteliali, nonché l&#39;effetto dei farmaci sulla differenziazione delle cellule epiteliali in generale. Originariamente sviluppato da Asselineau e Prunieras <sup&gt; 6 e modificato da Kopan et al. 7, il sistema di coltura organotipica epiteliale zattera è maturato in generale, modello di coltura relativamente facile, che coinvolge la crescita di cellule su collagene tappi mantenuta al di interfaccia aria-liquido (Figura 1A). Nel corso di 10-14 giorni, le cellule stratificare e differenziare, formando un epitelio pieno spessore che produce la differenziazione-specifica citocheratine. Zattere raccolto può essere esaminato istologicamente, nonché mediante tecniche standard molecolari e biochimiche. In questo articolo, si descrive un metodo per la generazione di colture zattera da cheratinociti umani primari. La stessa tecnica può essere utilizzata con linee cellulari epiteliali, e può essere facilmente adattato per l&#39;utilizzo con il tessuto epiteliale da biopsie normali o malati 8. Molti virus bersaglio sia l&#39;epitelio cutaneo o delle mucose, come parte del loro ciclo replicativo di vita. Nel corso degli ultimi anni, la possibilità di utilizzare cult zattera organotipicaURES come un metodo di studio virus-ospite interazioni cellulari è stato dimostrato per herpesvirus diverse, così come adenovirus, parvovirus, e poxvirus 9. Culture zattera organotipiche può quindi essere adattato per esaminare patogenesi virale, e sono gli unici mezzi per testare nuovi agenti antivirali per quei virus che non sono coltivabili in linee cellulari permanenti.

Watch this Scientific Journal Video about Generation of Organotypic Raft Cultures from Primary Human Keratinocytes at JoVE.com

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

Un<em&gt; In vitro</em&gt; Metodo per imitare<em&gt; In vivo</em&gt; Differenziazione epiteliale è descritto. Molti virus bersaglio le cellule epiteliali, come parte del loro ciclo vitale del virus, e questo metodo fornisce un mezzo di esaminare virus: interazioni host che più assomiglia quella che si verifica<em&gt; In vivo</em&gt;. Questa tecnica può essere utilizzata con cheratinociti primari, linee cellulari stabilizzate, nonché normale o tessuti malati biopsia.

Generazione di culture Raft organotipiche da cheratinociti umani primari

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

generation-organotypic-raft-cultures-from-primary-human

Research

JoVE Journal

Immunology and Infection

19.0K Views.  University of North Carolina-Chapel Hill. Un In vitro Metodo per imitare In vivo Differenziazione epiteliale è descritto. Molti virus bersaglio le cellule epiteliali, come parte del loro ciclo vitale del virus, e questo metodo fornisce un mezzo di esaminare virus: interazioni host che più assomiglia quella che si verifica In vivo. Questa tecnica può essere utilizzata con cheratinociti primari, linee cellulari stabilizzate, nonché normale o tessuti malati biopsia.

Video: Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Un metodo semplice ed efficace per isolare i macrofagi da Mixed colture primarie di cellule del fegato degli adulti

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

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Immunologia e infezioni

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

Generazione di cellule T regolatrici indotte da cellule primarie umane T naive e memoria

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

Isolamento primario Microglia misti da colture cellulari gliali del tessuto neonatale cervello di ratto

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

Generazione di Human alloantigene-specifiche cellule T da sangue periferico

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Generazione di ricombinante umano IgG anticorpi monoclonali da immortalizzate cellule ordinati B

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Preparation and Applications of Organotypic Thymic Slice Cultures

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell repertoire. In vitro models to study T lineage commitment and development have provided valuable insights into this process. However, these systems lack the complete three-dimensional thymic milieu necessary for T cell development and, therefore, are incomplete approximations of in vivo thymic selection. Some of the challenges related to modeling T cell development can be overcome by using in situ models that provide an intact thymic microenvironment that fully supports thymic selection of developing T cells. Thymic slice organotypic cultures complement existing in situ techniques. Thymic slices preserve the integrity of the thymic cortical and medullary regions and provide a platform to study development of overlaid thymocytes of a defined developmental stage or of endogenous T cells within a mature thymic microenvironment. Given the ability to generate ~20 slices per mouse, thymic slices present a unique advantage in terms of scalability for high throughput experiments. Further, the relative ease in generating thymic slices and potential to overlay different thymic subsets or other cell populations from diverse genetic backgrounds enhances the versatility of this method. Here we describe a protocol for the preparation of thymic slices, isolation and overlay of thymocytes, and dissociation of thymic slices for flow cytometric analysis. This system can also be adapted to study non-conventional T cell development as well as visualize thymocyte migration, thymocyte-stromal cell interactions, and TCR signals associated with thymic selection by two-photon microscopy.

We describe the preparation of thymic slices that, in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. Thymic slices can also be adapted for the in situ analysis of thymocyte migration, localization, and signaling via immunofluorescence and two-photon microscopy.

Preparazione e applicazioni di colture organotipiche timica Slice

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Generazione di derivate da monociti cellule dendritiche umane da sangue intero

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

L&#39;induzione di una risposta infiammatoria in colture primarie epatociti di topi

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

Piccolo RNA trasfezione in cellule primarie Th17 Human Next Generation elettroporazione

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Generazione di anticorpi monoclonali umani discriminante da rari B antigene-specifiche cellule circolanti nel sangue

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...