DRW è sostenuto da sovvenzioni NIH AI089972 e AI113217, e dalla mucosa Immunologia Studi della squadra, e in possesso di un Premio alla Carriera per medici scienziati del Wellcome Fondo Burroughs.

NOTA: Tutti gli esperimenti descritti erano in conformità con le Istituzionale Cura e uso Comitato Animal (IACUC) le linee guida e approvato dal Hospital Children Research animali (ARCH), Boston, Mass. 1. Preparazione dei reagenti <ol><li> 1,000x IL-4 Stock: Diluire citochina IL-4 a 20 pg / ml in H 2 O o 0,1% di siero albumina bovina (BSA). Distribuire in 200 aliquote microlitri provette da 1,5 ml microcentrifuga e conservare a -20 ° C. </li><li> 1,000x anti-CD40: ottenere dal produttore a 1,0 mg / ml, conservare a 4 ° C. </li><li> B stimolazione cellulare multimediale: per 425 ml di terreno RPMI-1640, aggiungere 75 ml di siero di vitello fetale appena scongelati (FCS), 10 ml di 1,0 M tampone HEPES, 5 ml di L-glutammina magazzino, 5 ml penicillina: streptomicina magazzino, 5 ml essenziali media non essenziali Aminoacidi microlitri minimi (MEM-NEAA), e 3,5 di 2-mercaptoetanolo. Tenere i supporti a 4 ° C per un massimo di una settimana. Aggiungere IL-4 (20 ng / ml) e anti-CD40 (1,0 mcg / ml) immediatamente prima dell&#39;usosolo il volume di supporti necessari. </li><li> 2% FCS-FACS Buffer: Aggiungi 10 ml di FCS a 490 ml di 1x PBS. Conservare a 4 ° C. </li><li> 2x soluzione tripsina-EDTA: Diluire 5 ml di soluzione di 10x tripsina-EDTA magazzino (5,0 g tripsina, EDTA 2,0 g per litro) in 20 ml di PBS 1x. Conservare tripsina-EDTA in 2x aliquote di lavoro a 4 ° C. Lasciar riscaldare a temperatura ambiente prima dell&#39;uso. </li><li> Siero fetale di vitello: Mantenere FCS scongelati a 4 ° C per un massimo di due settimane. Mettere in ghiaccio 15 minuti prima che la procedura di colorazione citoplasmatica. </li><li> Neutral-buffered formalina: Preparare una soluzione di formalina al 10% (3,7% paraformaldeide). Formalina è tossica e quindi gestirlo sotto una cappa di laboratorio mentre indossa appropriate DPI. Conservare formalina a temperatura ambiente in un armadietto di immagazzinaggio chimico. </li><li> Metanolo: Conservare metanolo assoluto (100%) a -20 ° C finché le cellule sono pronte per essere permeabilizzate. Si noti che il metanolo è infiammabile e deve essere conservato in un freezer adeguato. </li></ol> 2.Purificazione e attivazione delle cellule B <ol><li> Da una milza del mouse appena raccolte, preparare una sospensione di cellule singole premendo delicatamente l&#39;organo attraverso un colino cella di 70 micron con lo stantuffo di una siringa da 3 ml. In una provetta da centrifuga da 15 ml conica, raccogliere le cellule in 10 ml di PBS 1x e centrifugare a 300 xg per 5 minuti a 4 ° C. </li><li> Decantare il surnatante e risospendere pellet in 1 ml di globuli rossi Lysing Buffer per 5 min. Neutralizzare con 13,5 ml di 1x PBS e centrifugare a 300 xg per 5 min. </li><li> (Opzionale) magneticamente separare le cellule B e che utilizzano anti-CD45R (B220) -labeled perline, seguendo il protocollo di purificazione MACS secondo le istruzioni del produttore. </li><li> Stimolare le cellule B purificate a 1,0 x 10 6 cellule / ml in scaldato mezzi stimolazione delle cellule B contenente IL-4 (20 ng / ml) e anti-CD40 (1,0 mcg / ml) per 5 giorni a 37 ° C. Iniziare con 8 ml di coltura divisi in 2 pozzetti di una piastra di coltura da 6 pozzetti (4 ml). </li><li> Controllare culturi giornaliero di mantenere a 1,0 x 10 6 cellule / ml. Regolare la concentrazione suddividendo in altri pozzi e diluendo la cultura dei media con la stimolazione delle cellule B contenente IL-4 freschi e anti-CD40. </li></ol> 3. tripsinizzazione, Fissazione e permeabilizzazione <ol><li> Tripsinizzazione <ol><li> Raccogliere fino a 1.0 x 10 7 cellule coltivate in una provetta conica da 15 ml, centrifugare a 300 xg per 5 min, e decantare il surnatante. </li><li> Lavare con 10 ml di PBS per rimuovere la proteina residua dal supporto di raccolta delle cellule. Ripetere la fase di centrifugazione e decantare il surnatante. </li><li> Risospendere il pellet di cellule nel volume residuo dopo decantazione (circa 100 ml). </li><li> Aggiungere con cautela 800 ml di temperatura ambiente 0,1% (2x) tripsina-EDTA alle cellule. Versare lentamente con un angolo di 45 ° lungo la parete del tubo conico. </li><li> Chiudere la provetta e miscelare delicatamente inclinando il tubo in modo che il liquido corre lungo la lunghezza del tubo di tempo 5-6s. A filante, precipitato bianco potrebbe formarsi nella soluzione. Limitare il tempo tripsinizzazione di sotto di un min. </li><li> Placare la reazione tripsinizzazione con 1 ml di FCS freddo. Diluire il FCS immediatamente aggiungendo 10 ml di PBS freddo. </li><li> Centrifugare a 300 xg per 5 minuti a 4 ° C, poi decantare il supernatante e restituire i tubi in ghiaccio. </li></ol></li><li> Fissazione <ol><li> Risospendere le cellule nel volume residuo dopo decantazione. Se necessario, portare il volume fino a 100 ml con PBS freddo. </li><li> In una cappa aspirante, aggiungere 800 ml di soluzione di formalina tamponata neutra al 10% per le cellule e pipettare su e giù. Il tubo scalderà seguito all&#39;aggiunta di formalina. Incubare a 37 ° C per 10 min. </li></ol></li><li> Permeabilization <ol><li> Impostare un mixer da banco vortex ad una velocità di agitazione delicata. </li><li> Disegna 9 ml di freddo (-20 ° C), metanolo in un 10 ml pipetta sierologica. </li><li> Tenere la provetta contenente le cellule sopra il vortex e iniziare dolcemente shaking cellule. </li><li> Aggiungere metanolo freddo al tubo in modo cade a gocce sulle cellule mentre il tubo è ancora scuotendo modo che il metanolo è continuamente miscelazione. Dopo i primi 2 ml di metanolo la velocità di erogazione può essere aumentata a un flusso lento. </li><li> Lasciate che il tubo di sedersi sul ghiaccio per 30 minuti per completare la permeabilizzazione. In alternativa, le cellule possono essere conservati a -20 ° C per un mese. </li></ol></li></ol> 4. fluorescenza colorazione per IgG1 e IgE di conversione <ol><li> Campione Centrifugare a 300 xg per 5 minuti e decantare la soluzione di metanolo. Un precipitato bianco può sedimentare le cellule. </li><li> Aggiungere 10 ml di temperatura ambiente 1x PBS e centrifugare a 300 xg per 5 minuti per lavare le cellule. Ripetere una volta. Nota: Lavare il pellet con temperatura ambiente PBS dissolve precipitato in più che può formare dopo la fase 4.1. </li><li> Eseguire un lavaggio supplementare con 10 ml di 2% FCS-tampone FACS. </li><li> Distribuire campioni ad un 96-ben piatto rotondo fondoe centrifugare a 300 xg per 5 min. Decantare il surnatante e mettere il piatto su ghiaccio. </li><li> Stain in 60 ml di 2% di buffer FCS-FACS per bene con le seguenti diluizioni degli anticorpi: anti-IgM RPE / Cy7, 1: 200, anti-IgG1 PE, 1: 600, anti-IgE FITC, 1: 100, Anti- CD45R (B220) PerCP-Cy5.5, 1: 200 </li><li> Dopo 5 min di colorazione, lavare la piastra con 150 ml di 2% FCS-tampone FACS e centrifugare per 5 min a 300 x g. </li><li> Risospendere in 400 ml di 2% di buffer FCS-FACS e pipetta attraverso una 40 micron colino cella nylon in 5 ml FACS tubo. </li></ol> 5. Utilizzare la seguente strategia Gating: <ol><li> Isolare CD45R (B220) cellule positive da parte della popolazione brillamento linfociti situato sul / plot SSC FSC (Figura 1). </li><li> Dalle cellule positive B220, utilizzare la trama IgE e IgG1 di rivelare le popolazioni corrispondenti. </li></ol>

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The authors have no disclosures.

La stimolazione di topo milza cellule B in coltura con anti-CD40 e IL-4 simulerà T helper di tipo 2 (T H 2) le interazioni, il passaggio di classe incoraggiante IgG1 e IgE 5. Cellule B possono essere attivate per la RSI nel contesto di splenociti totale 12 o come purificati cellule B della milza 11. Come indicato nel protocollo (passo 2.3), B arricchimento delle cellule è opzionale, ed è a discrezione dello sperimentatore per determinare se sarebbe vantaggioso. Positivo sep...

Durante immunoglobuline catena pesante (IgH) ricombinazione switch di classe (CSR) in topi e nell&#39;uomo, il IgH μ esoni regione costanti (Cu) vengono cancellati e sostituiti da uno dei diversi gruppi di downstream esoni regione costanti (C H s) (es Cγ, Cε e Cα), risultando in un interruttore dalla produzione di IgM alla produzione di altre classi di Ig (ad esempio IgG, IgE, o IgA). CSR avviene entro interruttore (S) le regioni, che sono 1-10 sequenze kb situati 5 &#39;per ogni set di C H 1 s. L&#39;attivazione indotta citidina deaminasi (AID) enzima avvia CSR attraverso l&#39;attività citidina deamination. IgE è un mediatore chiave della malattia allergica 2 e una maggiore comprensione di come IgE è prodotto e regolato può aprire le porte a nuovi approcci terapeutici per la malattia atopica. In topi e nell&#39;uomo, IgE è isotipo Ig più strettamente regolata. IgE è normalmente rilevato livelli migliaia di times meno di altri IgH isotipi 3, ma possono essere molto elevati negli stati di malattia 4. Tuttavia, la RSI per IgE non è completamente comprensibile. In vitro attivazione delle cellule B con IL-4 in combinazione con anti-CD40 o LPS, induce la RSI sia IgG1 e IgE 5. Cellule B attivate esprimono il recettore a bassa affinità IgE FcεRII / CD23 2,6, che lega IgE solubile secreta nella cultura dopo CSR. Perciò quando si analizzano con citometria a flusso, le cellule B con il recettore legato IgE macchia simile a B cellule che esprimono endogenamente IgE 7. Mentre è noto che il mouse B cellule esprimono il recettore a bassa affinità IgG FcγRIIB1 (CD32) 8, nella nostra esperienza non sembra interferire con la misura di commutazione classe IgG1. Tuttavia, quando si misura IgE commutazione dopo l&#39;attivazione delle cellule B nella cultura, non specifica di superficie legato IgE può oscurare l&#39;analisi. Con i metodi di colorazione comuni, le cellule non-IgE-esprimono macchia positivo per IgE. Delineato qui è una strategia che è stata utilizzata per condurre test di CSR e rilevare vere cellule-IgE esprimono B da topi 9-11. Il trattamento delle cellule B attivate con tripsina, un reagente di laboratorio comune a digerire le proteine, rimuove sia cytophylic e la membrana superficiale legata IgE. Permeabilizzazione successiva e colorazione per citoplasmatica IgE rivela così le vere cellule-IgE che producono.

<table border="0" cellpadding="0" cellspacing="0" width="958">
	<tbody>
		<tr height="37">
			<td height="37" style="height:37px;width:301px;">Name</td>
			<td style="width:179px;">Company</td>
			<td style="width:135px;">Catalog Number</td>
			<td style="width:343px;">Comments</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Anti-Mouse IgE FITC</td>
			<td>BD Pharmingen</td>
			<td>553415</td>
			<td>clone R35-72</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Rat Anti-Mouse IgG1 PE</td>
			<td>BD Pharmingen</td>
			<td>550083</td>
			<td>clone A85-1</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Methanol</td>
			<td>BDH</td>
			<td>BDH1135-4LP</td>
			<td>Keep at -20 &#176;C</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Falcon Cell Strainer 40 &#181;m Nylon</td>
			<td>Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Falcon Cell Strainer 70 &#181;m Nylon</td>
			<td>Corning</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Anti-Hu/Mo CD45R(B220) PerCP-Cy5.5</td>
			<td>eBioscience</td>
			<td>45-4052-82</td>
			<td>clone RA3-6B2</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">anti-Mouse/Rat CD40</td>
			<td>eBioscience</td>
			<td>16-0402-85</td>
			<td>clone HM40-3</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">RPMI Medium 1640</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">HEPES (1M)</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">MEM-NEAA (100X)</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Phosphate Buffered Saline (10X)</td>
			<td>Lifetechnologies (Corning)</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr height="66">
			<td height="66" style="height:67px;">MACS CD45R (B220) microbeads&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-049-501</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">MACS purification column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">IL-4</td>
			<td>PeproTech</td>
			<td>214-14</td>
			<td>Reconstitute in water or 0.1% BSA</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Formalin Solution, Neutral Buffered, 10%</td>
			<td>Sigma Aldrich</td>
			<td>HT501128-4L</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Trypsin-EDTA Solution (10X)</td>
			<td>Sigma Aldrich</td>
			<td>T4174-100mL</td>
			<td>5.0g Trypsin, 2.0g EDTA&#376;4Na per Liter of 0.9% NaCl</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Penicillin-Streptomycin</td>
			<td>Sigma Aldrich</td>
			<td>P0781-100mL</td>
			<td>10,000U Penicillin; 10 mg/mL Streptomycin (100X)</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">L-Glutamine 200mM</td>
			<td>Sigma Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">2-mercaptoethanol (99%)</td>
			<td>Sigma Aldrich</td>
			<td>M6250-100mL</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Red cell lysis buffer</td>
			<td>Sigma Aldrich</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Rat Anti-Mouse IgM RPE/cy7</td>
			<td>SouthernBiotech</td>
			<td>1140-17</td>
			<td>clone 1B4B1</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">HyClone Fetal Calf Serum</td>
			<td>Thermo</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr height="161">
			<td height="161" style="height:161px;">B cell Stimulation media</td>
			<td></td>
			<td></td>
			<td style="width:343px;">B cell stimulation media consists of RPMI with fetal calf serum (15%), 20 mM HEPES, 1X MEM-NEAA, 2.0mM L-glutamine,&#160; 1X Penicillin-Streptomycin (Penicillin:100 U/ml, Streptomycin: 100 &#181;g/ml), Beta-mercaptoethanol (7 &#181;L/L), IL-4 (20 ng/mL), and anti-CD40 (1 &#181;g/mL)</td>
		</tr>
	</tbody>
</table>

detection of true ige-expressing mouse b lineage cells

B linfociti immunoglobuline catena pesante (IgH) ricombinazione switch di classe (CSR) è un processo in cui inizialmente espresso IgM passa ad altri isotipi IgH, come IgA, IgE e IgG. Misurazione di IgH CSR in vitro è un metodo fondamentale per lo studio di un certo numero di processi biologici che vanno dalla ricombinazione e riparazione del DNA agli aspetti della immunologia molecolare e cellulare. Test in vitro CSR coinvolge citofluorimetrica superficie di misurazione espressione Ig sulle cellule B attivate. Mentre la misurazione di IgA e IgG sottoclassi è semplice, misurazione delle IgE con questo metodo è problematico a causa IgE solubile legame FcεRII / CD23 espresso sulla superficie delle cellule B attivate. Qui si descrive una procedura unica per la misurazione accurata delle IgE-produzione di cellule del mouse B che hanno subito la RSI nella cultura. Il metodo si basa sulla tripsina-mediata scissione di complessi IgE-CD23 sulla superficie delle cellule, consente il rilevamento di IgE-cellule produttrici B lineage da cycolorazione toplasmic. Questa procedura offre una soluzione conveniente per l&#39;analisi di citometria di flusso della RSI a IgE.

Questa procedura è stata implementata con successo per studiare la RSI a IgE in cellule del mouse B. Per dimostrare l&#39;efficienza di misura della RSI, abbiamo stimolato le cellule B della milza di topo con anti-CD40 e IL-4 come descritto in precedenza 11. Dopo cinque giorni di stimolazione, le cellule sono state raccolte ed elaborate utilizzando il protocollo sopra descritto e colorati con fluorescenza marcata IgM, IgG1, IgE, gli anticorpi e B220 (CD45R). Gated sui linfociti sabbiatura (figura 1),...

Watch this Scientific Journal Video about Detection of True IgE-expressing Mouse B Lineage Cells at JoVE.com

Detection of True IgE-expressing Mouse B Lineage Cells

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 ...

detection-of-true-ige-expressing-mouse-b-lineage-cells

Research

JoVE Journal

Immunology and Infection

10.8K Views.  Brigham and Women's Hospital and Harvard Medical School. 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.

Video: Detection of True IgE-expressing Mouse B Lineage 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

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

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 immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching 1, 2. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

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 ...

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

Produzione ricombinante retrovirali e infezione delle cellule B

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Analisi rapida di aberrazioni cromosomiche in linfociti B mouse da PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

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, ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

Rilevazione rapida molecolare e la differenziazione dei virus influenzali A e B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

Rilevamento e l&#39;arricchimento di Rare antigene-specifiche cellule B per l&#39;analisi del fenotipo e funzione

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Rilevamento di attivazione Inflammasome e morte delle cellule Pyroptotic in macrofagi murini derivate da midollo osseo

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Identificazione e isolamento di Oligopotent e dei progenitori mieloidi Lineage-commesso dal midollo osseo del topo

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1&#8211;2 copies) integration using inverse nested PCR.

Rilevamento di copia basso numero di DNA virale integrato formato da In Vitro l'infezione di epatite B

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...