Siamo grati al laboratorio Martin per utili discussioni. Questa pubblicazione è sostenuta da una sovvenzione da parte del Canadian Institute of Health Research (MOP-89783). AM è supportata da un Canada Research Chair Premio Tier II.

Fase I: isolamento delle cellule B della milza attraverso l&#39;arricchimento magnetico <ol><li> Topi sperimentali devono essere di età compresa tra 8-12 settimane per garantire la piena maturazione del sistema immunitario. </li><li> Euthanize il mouse dislocazione cervicale e immergersi in etanolo al 70%. Rimuovere chirurgicamente la milza facendo un&#39;incisione attraverso la pelle ei muscoli del ipocondrio sinistro dell&#39;addome e tagliata in tre pezzi grossi in tampone fosfato (PBS). Garantire tutti gli strumenti e reagenti sono sterili. </li><li> Utilizzando la fine di gomma di una siringa da 1 ml stantuffo, poltiglia delicatamente la milza attraverso un filtro 70 micron cellula in PBS. Assicuratevi di utilizzare un movimento di spinta piuttosto che un movimento di rettifica rettifica può portare alla rottura dei più grandi (cioè proliferanti) cellule. </li><li> Spin giù le cellule a non più di 350 g per 5 minuti e risospendere il pellet in PBS. Contare le cellule risospese con un emocitometro. </li><li> Preparare una sospensione di 1x10 8 cellule / ml in PBS con 5% di siero di ratto normale in un tubo sterile 5 ml in polistirolo con un tappo (volume massimo di 2 ml per provetta). </li><li> Aggiungere 50 ml di EasySep negativa cocktail mouse selezione arricchimento cella B per ogni ml di cellule. Pipetta la miscela, tappo i tubi, e mettere in frigo per 15 minuti. </li><li> Aggiungere 100 ul EasySep Cocktail selezione biotina per ogni ml di cellule. Pipetta la miscela, tappo i tubi, e mettere in frigo per 15 minuti. </li><li> Aggiungere 100 ml di EasySep nanoparticelle magnetiche (garantire le nanoparticelle sono mescolati bene e la soluzione è omogenea) per ogni ml di cellule. Pipetta la miscela, tappo i tubi, e mettere in frigo per 5 minuti. </li><li> Top la soluzione a 2,5 ml con PBS e collocare in un magnete EasySep per 5 minuti. Invertire il magnete e tubo e versare subito in una provetta di polistirolo nuovo. Non agitare il tubo come cellule negativamente selezionati verranno magneticamente legato alle pareti del tubo. </li><li> Per aumentare ulteriormente la purezza della sospensione cellulare, il tubo può essere reinserito nel magnete per altri 5 minuti e versato in un nuovo tubo. Questo può ridurre il recupero complessivo delle cellule. </li><li> Purezza delle cellule B può essere valutata mediante citometria a flusso dei marcatori di superficie delle cellule B. A seguito di arricchimento, abbiamo sempre vedere una purezza&gt; 95% di cellule positive B220. </li></ol> Fase II: colorazione della cellula CFSE <ol><li> Risospendere le cellule isolate in caldo PBS integrato con 0,1% di albumina sierica bovina ad una concentrazione finale di 1 x 10 6 cellule per ml. </li><li> Preparare una soluzione stock di 5mM CFSE (diluita in sterili dimetilsolfossido) e aggiungere 2μL per ogni ml di cellule nel tubo. La concentrazione finale di 10μM è ottimale per la colorazione delle cellule B primarie. </li><li> Incubare a 37 ° C per 10 minuti al buio (nota: CFSE è sensibile alla luce e deve essere conservato al buio, quando possibile). </li><li> Spegnere la macchia con l&#39;aggiunta di un uguale volume di siero di vitello bovini verso le cellule e di incubazione in ghiaccio per 5 minuti. </li><li> Lavare le cellule attraverso un ricorso supplementare i tubi con terreno di coltura (vedi punto III per ricetta) e centrifugazione a 350 g. Si noti che almeno 5 volte l&#39;equivalente volume di mezzi di comunicazione deve essere aggiunto per contrastare la viscosità del siero di vitello e bovino per le cellule di produrre un pellet. </li><li> Lavare le cellule due volte di più nel terreno di coltura. Le cellule sono ora pronti per la stimolazione. </li></ol> Fase III: la stimolazione delle cellule con LPS <ol><li> Preparare il supporto in coltura in vitro delle cellule B, completandola RPMI 1640 con il 10% di siero fetale bovino e 50 mM β-mercaptoetanolo /. </li><li> Preparare il supporto stimolazione con l&#39;aggiunta di LPS ad una concentrazione finale di 50μg/mL. Aliquota 125 ml di mezzo di stimolazione in ciascun pozzetto di un fondo piatto da 96 pozzetti piastra di coltura dei tessuti. </li><li> Preparare il CFSE macchiato di linfociti B nel terreno di coltura senza LPS ad una concentrazione finale di 3,2 x 10 6 cellule / ml. Aggiungere 125 ml di sospensione cellulare ai pozzetti contenenti i media stimolazione e mescolare pipettando delicatamente. </li><li> Ogni ora contiene ben 4 x 10 5 cellule in una concentrazione finale LPS di 25 mg / ml. Ciò fornisce livelli ottimali di proliferazione delle cellule e il passaggio di classe, anche se le modifiche a questi valori possono essere fatte come desiderato. </li><li> Incubare le cellule a 37 ° C e 5% di CO 2 per 72 a 96 ore. Dopo 48 ore, gruppi di grandi cellule B proliferanti sarà chiaramente visibile al microscopio. </li></ol> Fase IV: L&#39;analisi citofluorimetrica di switching di classe <ol><li> Quando si è pronti a guardare il passaggio di classe, rimuovere le cellule dal loro condizioni di coltura e lavarli due volte in 1 ml di tampone colorazione (PBS con il 2% di siero bovino di vitello). </li><li> Per evitare che non specifica colorazione anticorpale delle cellule, agglomerare le cellule facendo girare giù a 350 g e incubate in 100μL di buffer di colorazione con 5% di siero normale mouse e 1μg di FcBlock per106 cellule per 15 minuti in ghiaccio. Il blocco delle cellule deve essere eseguita su ghiaccio. </li><li> Senza lavarsi le cellule, aggiungere 1μg per 10 6 cellule di un tag fluorescente contro topo IgG3 anticorpi e permettono alle cellule di macchia per 30 minuti in ghiaccio. </li><li> Lavare le cellule due volte tramite centrifugazione e risospendere in 200-300 ml di buffer di colorazione. Le cellule sono ora pronti per la valutazione in citometria a flusso. </li><li> CFSE è ottimale eccitata a 492 nm (da un laser a ioni argon) ed emette a 517 nm. Lo spettro di eccitazione ed emissione dell&#39;anticorpo IgG3 dipende dalla sua coniugato con il colorante fluorescente. </li></ol> Rappresentante Risultati A seguito di arricchimento magnetica, la sospensione cellulare dovrebbe apparire come una popolazione omogenea di cellule indistinguibili piccoli circolare (Figura 1A). Dopo 48-72 ore di stimolazione, gruppi isolati di cellule proliferanti allargata sarà chiaramente visibile (Figura 1B). Gran parte delle cellule non proliferanti apparirà piccole e granulari come apoptosi. RSI può normalmente essere rilevata ai massimi livelli tra 72 e 96 ore dopo la stimolazione prima che la maggior parte delle cellule cominciano a morire 7. In questa fase, le cellule devono avere subito una serie di divisioni cellulari con cellule acceso che appaiono nella popolazione dopo cellula figlia. Un diagramma di flusso rappresentante citometria di diluizione CFSE e espressione di superficie di IgG3 dopo 96 ore di stimolazione LPS può essere visto in Figura 2. Tabella 1. Isotipo commutazione cocktail. Nota: valori di concentrazione stimolanti sono solo raccomandazioni. Titolazioni può essere necessario. <table border="1"><tbody><tr><td> Isotipo desiderato </td><td> Stimolazione Cocktail </td></tr><tr><td> IgG1and IgE </td><td> LPS (25μg/mL) e IL-4 (10 ng / ml) </td></tr><tr><td> IgG1and IgE </td><td> Anti-CD40 (10μg/mL) e IL-4 (10 ng / ml) </td></tr><tr><td> IgG2a </td><td> LPS (25μg/mL) e IFN-γ (10 ng / ml) </td></tr><tr><td> IgG2b e IgG3 </td><td> LPS (25μg/mL) </td></tr><tr><td> IgA </td><td> LPS (25μg/mL), TGF-β (2 ng / ml) e IL-5 (1.5 ng / ml) </td></tr></tbody></table><img src="/files/ftp_upload/2130/2130fig1.jpg" alt="Figura 1" /> Figura 1. Isolamento LPS e la stimolazione delle cellule B della milza. (A) magneticamente arricchito cellule B della milza prima della stimolazione LPS. (B) 48 ore dopo la stimolazione con 25 mg / ml di LPS. Le cellule proliferanti sono visti come i cluster in una di sfondo delle celle sabbiatura e apoptosi. <img src="/files/ftp_upload/2130/2130fig2.jpg" alt="Figura 2" /> Figura 2. Proliferazione e di passare ricombinazione classe dopo 96 ore. (A) diluizione CFSE per LPS stimola le cellule dopo 96 ore di cultura. Ogni picco indipendente rappresenta una diluizione della fluorescenza CFSE corrispondente ad una divisione cellulare indipendente. (B) l&#39;espressione IgG3 mostrano l&#39;emergere di una popolazione IgG3 positivo dopo diversi cicli di divisione cellulare.

<ol>
	<li>Hodgkin, P. D., Lee, J. H., Lyons, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cell+differentiation+and+isotype+switching+is+related+to+division+cycle+number.">B cell differentiation and isotype switching is related to division cycle number.</a> J Exp Med. 184, 277-281 (1996).</li><li>McCall, M. N., Hodgkin, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Switch+recombination+and+germ-line+transcription+are+division-regulated+events+in+B+lymphocytes.">Switch recombination and germ-line transcription are division-regulated events in B lymphocytes.</a> Biochim Biophys Acta. 1447, 43-50 (1999).</li><li>Manis, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=53BP1+links+DNA+damage-response+pathways+to+immunoglobulin+heavy+chain+class-switch+recombination.">53BP1 links DNA damage-response pathways to immunoglobulin heavy chain class-switch recombination.</a> Nat Immunol. 5, 481-487 (2004).</li><li>de Yebenes, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-181b+negatively+regulates+activation-induced+cytidine+deaminase+in+B+cells.">miR-181b negatively regulates activation-induced cytidine deaminase in B cells.</a> J Exp Med. 205, 2199-2206 (2008).</li><li>McBride, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+class+switch+recombination+and+somatic+mutation+by+AID+phosphorylation.">Regulation of class switch recombination and somatic mutation by AID phosphorylation.</a> J Exp Med. 205, 2585-2594 (2008).</li><li>Ramachandran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNF8/RNF168+ubiquitin+ligase+cascade+facilitates+class+switch+recombination.">The RNF8/RNF168 ubiquitin ligase cascade facilitates class switch recombination.</a> Proc Natl Acad Sci U S A. 107, 809-8014 (2010).</li><li>Zaheen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AID+constrains+germinal+center+size+by+rendering+B+cells+susceptible+to+apoptosis.">AID constrains germinal center size by rendering B cells susceptible to apoptosis.</a> Blood. 114, 547-554 (2009).</li><li>Chaudhuri, J., Alt, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Class-switch+recombination:+interplay+of+transcription,+DNA+deamination+and+DNA+repair.">Class-switch recombination: interplay of transcription, DNA deamination and DNA repair.</a> Nat Rev Immunol. 4, 541-552 (2004).</li><li>Kaminski, D. A., Stavnezer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimuli+that+enhance+IgA+class+switching+increase+histone+3+acetylation+at+S+alpha,+but+poorly+stimulate+sequential+switching+from+IgG2b.">Stimuli that enhance IgA class switching increase histone 3 acetylation at S alpha, but poorly stimulate sequential switching from IgG2b.</a> Eur J Immunol. 37, 240-251 (2007).</li><li>Kaminski, D. A., Stavnezer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+class+switching+differs+among+SJL,+C57BL/6+and+129+mice.">Antibody class switching differs among SJL, C57BL/6 and 129 mice.</a> Int Immunol. 19, 545-556 (2007).</li></ol>

Nessun conflitto di interessi dichiarati.

Il protocollo di cui sopra fornisce un test standard per analizzare l&#39;espressione AID e funzione durante la commutazione di classe delle primarie cellule B murini. Questo protocollo utilizza LPS batterici per indurre il passaggio da IgM a IgG3, anche se le modifiche ai mezzi di stimolazione può essere fatto per indurre il passaggio ad altri isotipi (riassunti nella Tabella 1 8, 9). Abbiamo notato che, per ragioni ancora sconosciute, siero di vitello fetale possono influenzare...

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		<th>Comment</th>
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<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14190</td>
<td>&nbsp;</td>
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<td>EasySep Mouse B Cell Enrichment Kit</td>
<td>&nbsp;</td>
<td>Stemcell Technologies</td>
<td>19754</td>
<td>&nbsp;</td>
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<td>Polystyrene tubes</td>
<td>&nbsp;</td>
<td>Becton Dickinson Falcon</td>
<td>352058</td>
<td>&nbsp;</td>
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<td>Bovine Serum Albumin</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>A7030</td>
<td>&nbsp;</td>
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<td>CellTrace CFSE Cell Proliferation Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>C34554</td>
<td>&nbsp;</td>
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<td>Bovine Calf Serum</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>SH30072.03</td>
<td>&nbsp;</td>
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<td>RPMI 1640 Culture Medium</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11875</td>
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<td>Lipopolysaccharides from Escherichia coli</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>L6529</td>
<td>&nbsp;</td>
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<td>&beta;-mercaptoethanol</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>21985</td>
<td>&nbsp;</td>
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<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>SH30396.03</td>
<td>&nbsp;</td>
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<td>Normal Mouse Serum</td>
<td>&nbsp;</td>
<td>Jackson ImmunoResearch</td>
<td>011-000</td>
<td>&nbsp;</td>
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<td>Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block)</td>
<td>&nbsp;</td>
<td>Becton Dickinson Pharmingen</td>
<td>553141</td>
<td>&nbsp;</td>
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<td>Rat Anti-Mouse IgG3</td>
<td>&nbsp;</td>
<td>Becton Dickinson Pharmingen</td>
<td>553401</td>
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induction and assessment of class switch recombination in purified murine b cells

L&#39;immunità umorale è il ramo del sistema immunitario mantenuto da parte delle cellule B e mediato attraverso la secrezione di anticorpi. Dopo l&#39;attivazione delle cellule B, il locus immunoglobuline subisce una serie di modificazioni genetiche per alterare la capacità di legame e le funzioni effettrici degli anticorpi secreti. Questo processo è evidenziato da un evento di ricombinazione genomica noto come ricombinazione di classe switch (CSR) in cui viene sostituito il valore predefinito di anticorpi IgM isotipo per una delle IgG, IgA o IgE. Ogni isotipo possiede diverse funzioni effettrici rendendo in tal modo la RSI fondamentale per il mantenimento dell&#39;immunità. Diversificazione del locus immunoglobuline è mediato dalla attivazione degli enzimi deaminasi indotta citidina (AID). Un video che descrive schematicamente questo processo in dettaglio è disponibile online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). Attività AID e la via della RSI sono comunemente studiate nella valutazione della funzione delle cellule B e immunità umorale nei topi. Il protocollo descritto in questo rapporto presenta un metodo di isolamento delle cellule B da milza murina e conseguente stimolazione con lipopolisaccaride batterico (LPS) per indurre il passaggio a classe IgG3 (per isotipi anticorpali altri vedi Tabella 1). Inoltre, la colorazione della cellula colorante fluorescente Carboxyfluorescein succinimidile estere (CFSE) è usato per controllare la divisione cellulare delle cellule stimolate, un processo cruciale per il passaggio isotipo 1, 2. La regolamentazione degli aiuti e il meccanismo con cui la RSI si sono ancora poco chiari, e quindi test in classe interruttore in vitro forniscono un metodo affidabile per testare questi processi in diversi modelli murini. Questi test sono stati utilizzati in precedenza nel contesto di carenza di gene utilizzando topi knockout 3. Inoltre, in vitro il passaggio delle cellule B può essere preceduto da trasduzione virale di modulare l&#39;espressione genica attraverso knockdown RNA o espressione del transgene 4-6. I dati da questi tipi di esperimenti hanno influenzato la nostra comprensione della attività AID, la risoluzione della reazione della RSI, e di anticorpi-mediata nel topo.

Watch this Scientific Journal Video about Induction and Assessment of Class Switch Recombination in Purified Murine B Cells at JoVE.com

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

A seguito di esposizione agli antigeni, sottopopolazioni di cellule B attivate subire un processo noto come ricombinazione interruttore classe (CSR) per la produzione di anticorpi isotipi con distinte funzioni effettrici. Il protocollo descritto in questo rapporto spiega come la RSI può essere indotta e analizzati<em&gt; In vitro</em&gt; Al fine di studiare la funzione delle cellule B.

Induzione e Valutazione della ricombinazione Classe Switch in depurata cellule B murini

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

induction-assessment-class-switch-recombination-purified-murine-b

Research

JoVE Journal

Immunology and Infection

18.0K Views.  University of Toronto. A seguito di esposizione agli antigeni, sottopopolazioni di cellule B attivate subire un processo noto come ricombinazione interruttore classe (CSR) per la produzione di anticorpi isotipi con distinte funzioni effettrici. Il protocollo descritto in questo rapporto spiega come la RSI può essere indotta e analizzati In vitro Al fine di studiare la funzione delle cellule B.

Video: Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Modello murino di CD40-attivazione delle cellule B

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Ricerca

Immunologia e infezioni

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

L&#39;identificazione del DNA Mutazioni in depurata ematopoietiche cellule staminali / progenitrici

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

Rilevazione di True IgE-esprimono mouse B Cells Lineage

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

Induzione di Murine infiammazione intestinale per trasferimento adottivo di Effector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alto</sup&gt; T Cells in topi immunodeficienti

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

The immunomodulatory properties of human mesenchymal stem cells (MSC) appear increasingly relevant for clinical application. Using a co-culture system of MSCs and peripheral blood leukocytes pre-stained with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE), we describe the in vitro assessment of MSC immunomodulation on effector leukocyte proliferation and specific subpopulations.

Valutazione della proprietà immunomodulanti di cellule staminali mesenchimali umane (MSC)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Un modello murino di Gruppo B<em&gt; Streptococcus</em&gt; Colonizzazione vaginale

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Genome-wide Analysis of HDAC Inhibitor-mediated Modulation of microRNAs and mRNAs in B Cells Induced to Undergo Class-switch DNA Recombination and Plasma Cell Differentiation

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA recombination (CSR), and plasma cell differentiation. In recent years, epigenetic modifications or factors, such as histone deacetylation and microRNAs (miRNAs), have been shown to interact with B-cell genetic programs to shape antibody responses, while the dysfunction of epigenetic factors has been found to lead to autoantibody responses. Analyzing genome-wide miRNA and mRNA expression in B cells in response to epigenetic modulators is important for understanding the epigenetic regulation of B-cell function and antibody response. Here, we demonstrate a protocol for inducing B cells to undergo CSR and plasma cell differentiation, treating these B cells with histone deacetylase (HDAC) inhibitors (HDIs), and analyzing mRNA and microRNA expression. In this protocol, we directly analyze complementary DNA (cDNA) sequences using next-generation mRNA sequencing (mRNA-seq) and miRNA-seq technologies, mapping of the sequencing reads to the genome, and quantitative reverse transcription (qRT)-PCR. With these approaches, we have defined that, in B cells induced to undergo CSR and plasma cell differentiation, HDI, an epigenetic regulator, selectively modulates miRNA and mRNA expression and alters CSR and plasma cell differentiation.

Epigenetic factors can interact with genetic programs to modulate gene expression and regulate B cell function. By combining in vitro B-cell stimulation, qRT-PCR, and high-throughput microRNA-sequence and mRNA-sequence approaches, we can analyze the epigenetic modulation of miRNA and gene expression in B cells.

Modulazione da analisi di inibitore HDAC genoma di microRNA e mRNAs in B le cellule indotte a sottoporsi switch classe DNA ricombinazione e differenziazione delle cellule di Plasma

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

In Vitro e In Vivo valutazione della attività soppressiva T, B e cellule mieloidi e risposte umorali dai destinatari del trapianto

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

Analisi di taglio indotta da flusso di migrazione delle cellule di B di zona marginale murino In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.