Wir sind dankbar für die Martin-Labor für hilfreiche Diskussionen. Diese Publikation wird durch einen Zuschuss von der Canadian Institute of Health Research (MOP-89783) unterstützt. AM wird durch einen Canada Research Chair Tier II-Award unterstützt.

Schritt I: Isolierung von Milz B-Zellen über magnetische Anreicherung <ol><li> Experimentelle Mäuse sollten im Alter von 8-12 Wochen in voller Reifung des Immunsystems zu gewährleisten. </li><li> Euthanize der Maus durch Genickbruch und genießen in 70% Ethanol. Chirurgisch entfernen Sie die Milz durch einen Einschnitt durch die Haut und Muskulatur der linken Hypochondrium des Bauches und schneiden Sie es in drei großen Stücken in Phosphat-gepufferter Kochsalzlösung (PBS). Sicherstellen, dass alle Werkzeuge und Reagenzien sind steril. </li><li> Mit dem Gummi Ende einer 1 ml Spritze Kolben, der Milz sanft Maische durch ein 70 um Zellsieb in PBS. Achten Sie darauf, eine schiebende Bewegung statt einer Schleifmaschine Bewegung als Schleifen zum Bruch von größeren (dh proliferierende) Zellen führen kann. </li><li> Spin down die Zellen bei nicht mehr als 350 g für 5 Minuten und das Pellet in PBS. Zählen Sie die resuspendierten Zellen mit einer Zählkammer. </li><li> Eine Suspension von 1x10 8 Zellen / ml in PBS mit 5% normalem Rattenserum in einer sterilen 5 ml Polystyrol Röhrchen mit einer Kappe (maximales Volumen von 2 ml pro Röhrchen). </li><li> Add 50 ul EasySep Negative Selection Maus B-Zell-Enrichment-Cocktail für jeden mL der Zellen. Pipette die Mischung, Kappe der Rohre, und in den Kühlschrank stellen für 15 Minuten. </li><li> 100 l EasySep Biotin Selection Cocktail für jeden mL der Zellen. Pipette die Mischung, Kappe der Rohre, und in den Kühlschrank stellen für 15 Minuten. </li><li> Add 100 L EasySep Magnetische Nanopartikel (sicher Nanopartikel sind gut gemischt und die Lösung homogen ist) für jeden mL der Zellen. Pipette die Mischung, Kappe der Rohre, und in den Kühlschrank stellen für 5 Minuten. </li><li> Top die Lösung bis 2,5 ml mit PBS und in einen EasySep Magnet für 5 Minuten. Kehren Sie die Magnet-und Rohr-und gießen schnell in eine neue Polystyrol-Röhrchen. Schütteln Sie die Röhre als negativ ausgewählten Zellen wird magnetisch auf der Rohrwandungen gebunden zu sein. </li><li> Zur weiteren Erhöhung der Reinheit der Zellsuspension kann das Rohr in den Magneten für weitere 5 Minuten wieder eingesetzt werden und in eine neue Röhre. Dies kann die gesamte Zelle Erholung. </li><li> B-Zell-Reinheit kann durch durchflusszytometrische Analyse der B-Zell-Oberflächenmarker beurteilt werden. Nach Anreicherung wir konsequent sehen einer Reinheit von&gt; 95% B220-positiven Zellen. </li></ol> Step II: CFSE Zellfärbung <ol><li> Resuspendieren der isolierten Zellen in warmen PBS mit 0,1% Rinderserumalbumin in einer Endkonzentration von 1 x 10 6 Zellen pro ml ergänzt. </li><li> Bereiten Sie einen 5mm Stammlösung von CFSE (verdünnt in sterile Dimethylsulfoxid) und fügen 2μL für jeden ml der Zellen in die Röhre. Die Endkonzentration von 10 &amp;mgr; m ist optimal für die Färbung von primären B-Zellen. </li><li> Bei 37 ° C für 10 Minuten im Dunkeln (Anmerkung: CFSE ist lichtempfindlich und sollte im Dunkeln, wenn möglich gehalten werden). </li><li> Quench der Fleck durch Zugabe eines gleichen Volumens von bovinem Kälberserum Ihre Zellen und Inkubation auf Eis für 5 Minuten. </li><li> Waschen Sie die Zellen im Rückgriff auf die Röhrchen mit Kulturmedium (siehe Schritt III für Rezept) und Zentrifugation bei 350 g. Beachten Sie, dass mindestens 5-mal das Volumen umgerechnet Medien hinzugefügt werden müssen, um die Viskosität des bovinem Kälberserum entgegenzuwirken und für die Zellen, um ein Pellet zu produzieren. </li><li> Waschen Sie die Zellen noch zweimal in Kulturmedium. Die Zellen sind nun bereit für die Stimulation. </li></ol> Step III: Cell Stimulation mit LPS <ol><li> Bereiten Sie Ihre in vitro B-Zell-Kulturmedium durch Ergänzung RPMI 1640 mit 10% fötalem Kälberserum und 50 uM β-Mercaptoethanol /. </li><li> Bereiten Sie Ihre Stimulation Medium durch Zugabe von LPS in einer Endkonzentration von 50μg/mL. Aliquot 125 ul der Stimulation Medium in jede Vertiefung einer Flachboden 96-Well-Gewebekulturplatten. </li><li> Bereiten Sie Ihre CFSE-gefärbte B-Zellen in Kulturmedium ohne LPS in einer Endkonzentration von 3,2 x 10 6 Zellen / ml. Add 125 ul der Zellsuspension in die Vertiefungen mit Ihrem Stimulation mittel-und Mix durch Pipettieren vorsichtig. </li><li> Jedes der gut enthält jetzt 4 x 10 5 Zellen in eine endgültige LPS Konzentration von 25 ug / mL. Dies sorgt für eine optimale Niveau der Zellproliferation und Klassenwechsel, obwohl Änderungen dieser Werte können aus beliebig. </li><li> Inkubieren Sie die Zellen bei 37 ° C und 5% CO 2 für 72 bis 96 Stunden. Nach 48 Stunden wird Cluster von großen proliferierenden B-Zellen deutlich sichtbar unter dem Mikroskop. </li></ol> Schritt IV: Durchflusszytometrische Analyse der Klassenwechsel <ol><li> Wenn Sie bereit sind, bei Klassenwechsel aussehen, entfernen Sie Ihre Zellen von ihrer Kultur Bedingungen und waschen Sie sie zweimal in 1 ml Färbepuffer (PBS mit 2% bovinem Kälberserum). </li><li> Um nicht-spezifische Antikörper-Färbung von Ihren Zellen, Pellet die Zellen durch Drehen sie sich bei 350 g und Gründerzentren verhindernte sie in 100 &amp;mgr; Färbepuffer mit 5% normalem Maus-Serum und 1 g des FcBlock per106 Zellen für 15 Minuten auf Eis. Zellen blockieren sollten auf Eis durchgeführt werden. </li><li> Ohne Waschen Sie Ihre Zellen, fügen 1 g pro 10 6 Zellen eines fluoreszenzmarkierten anti-Maus IgG3-Antikörper und damit die Zellen für 30 Minuten auf Eis Fleck. </li><li> Waschen Sie die Zellen zweimal durch Zentrifugieren und Resuspendieren in 200-300 ul Färbepuffer. Die Zellen sind nun bereit für die Beurteilung mittels Durchflusszytometrie. </li><li> CFSE ist optimal bei 492 nm angeregt (das von einem Argon-Ionen-Laser) und emittiert bei 517 nm. Die Anregungs-und Emissionsspektrum des IgG3-Antikörper ist abhängig von seiner Konjugat Fluoreszenzfarbstoff. </li></ol> Repräsentative Ergebnisse Nach magnetische Anreicherung, sollte die Zellsuspension wie eine homogene Population von ununterscheidbar kleine runde Zellen (Abbildung 1A) zu suchen. Nach 48-72 Stunden der Stimulation, wird isolierten Clustern von vergrößerten proliferierenden Zellen deutlich sichtbar (Abbildung 1B). Ein großer Anteil der nicht-proliferierenden Zellen erscheinen klein und körnig, wie sie Apoptose. CSR kann in der Regel auf der höchsten Ebene zwischen 72 und 96 Stunden nach Stimulation erkannt werden, bevor die meisten der Zellen bis 7 beginnen zu sterben. In diesem Stadium sollten die Zellen eine Reihe von Zellteilungen mit eingeschaltet Zellen, die in der späteren Tochter Zellpopulation unterzogen haben. Ein Vertreter durchflusszytometrischen Grundstück von CFSE Verdünnung und Oberflächenexpression von IgG3 nach 96 Stunden LPS-Stimulation ist in Abbildung 2 zu sehen. Tabelle 1. Isotyp Wechsel Cocktails. Hinweis: Stimulant Konzentration Werte sind nur Empfehlungen. Titrationen kann notwendig sein. <table border="1"><tbody><tr><td> Gewünschte Isotyp </td><td> Stimulation Cocktail </td></tr><tr><td> IgG1and IgE </td><td> LPS (25μg/mL) und IL-4 (10 ng / mL) </td></tr><tr><td> IgG1and IgE </td><td> Anti-CD40 (10μg/mL) und IL-4 (10 ng / mL) </td></tr><tr><td> IgG2a </td><td> LPS (25μg/mL) und IFN-γ (10 ng / mL) </td></tr><tr><td> IgG2b und IgG3 </td><td> LPS (25μg/mL) </td></tr><tr><td> IgA </td><td> LPS (25μg/mL), TGF-β (2 ng / ml) und IL-5 (1,5 ng / mL) </td></tr></tbody></table><img src="/files/ftp_upload/2130/2130fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Isolation und LPS-Stimulation von Milz-B-Zellen. (A) Magnetisch Milz B-Zellen vor LPS-Stimulation angereichert. (B) 48 Stunden nach Stimulation mit 25 pg / ml LPS. Zellen sind als Cluster in einem Hintergrund von Strahlen und apoptotische Zellen gesehen. <img src="/files/ftp_upload/2130/2130fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Proliferation und die Klasse wechseln Rekombination nach 96 Stunden. (A) CFSE Verdünnung für LPS stimulierten Zellen nach 96 Stunden in Kultur. Jeder unabhängige Peak repräsentiert eine Verdünnung in CFSE Fluoreszenz entspricht einem unabhängigen Zellteilung. (B) IgG3 Ausdruck zeigt die Entstehung einer IgG3 positive Bevölkerung nach mehreren Runden der Zellteilung.

<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>

Das Protokoll skizzierten bietet einen Standard-Test, um AID Expression und Funktion während des Unterrichts Schalten von primären murinen B-Zellen zu analysieren. Dieses Protokoll verwendet bakterielle LPS zu induzieren Wechsel von IgM zu IgG3, obwohl Änderungen an der Stimulation Medien gemacht zu veranlassen, auf andere Isotypen (zusammengefasst in Tabelle 1 8, 9). Wir haben festgestellt, dass für noch nicht bekannte Gründe, fetales Kälberserum kann das Niveau der Klasse ...

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<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14190</td>
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<td>EasySep Mouse B Cell Enrichment Kit</td>
<td>&nbsp;</td>
<td>Stemcell Technologies</td>
<td>19754</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>
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<td>CellTrace CFSE Cell Proliferation Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>C34554</td>
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<td>Bovine Calf Serum</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>SH30072.03</td>
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<td>&beta;-mercaptoethanol</td>
<td>&nbsp;</td>
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<td>21985</td>
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<td>&nbsp;</td>
<td>HyClone</td>
<td>SH30396.03</td>
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<td>Normal Mouse Serum</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>
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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

Humorale Immunität ist der Zweig des Immunsystems durch B-Zellen gehalten und vermittelt durch die Sekretion von Antikörpern. Bei B-Zell-Aktivierung, erfährt der Immunglobulin-Locus eine Reihe von genetischen Veränderungen, die Bindekapazität und Effektor-Funktion der sekretierten Antikörper verändern. Dieser Prozess wird durch eine genomische Rekombination Veranstaltung als Klasse-Schalter Rekombination (CSR), in denen die Standard-IgM-Antikörper-Isotyp für einen IgG, IgA, IgE oder substituierten bekannt ist hervorgehoben. Jeder Isotyp besitzt verschiedene Effektor-Funktionen wodurch CSR entscheidend für die Aufrechterhaltung der Immunität. Die Diversifizierung der Immunglobulin-Locus wird durch das Enzym activation-induced Cytidindeaminase (AID) vermittelt. Eine schematische Video beschreibt diesen Prozess im Detail ist online verfügbar (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID-Aktivität und der CSR-Weg werden häufig bei der Beurteilung der B-Zell-Funktion und humoralen Immunität in Mäusen untersucht. Das Protokoll in diesem Bericht beschriebenen präsentiert eine Methode der B-Zell-Isolierung von murinen Milz und nachfolgende Stimulation mit bakteriellem Lipopolysaccharid (LPS) zu induzieren Klasse Umstellung auf IgG3 (für andere Antikörper-Isotypen siehe Tabelle 1). Darüber hinaus ist das fluoreszierende Zellfärbung Farbstoff Carboxyfluorescein Succinimidylester (CFSE) verwendet werden, um die Zellteilung der stimulierten Zellen, ein Prozess von entscheidender Bedeutung für Schalt-1, 2 Isotyp zu überwachen. Die Regulierung der AID und der Mechanismus, durch den CSR auftritt, sind noch unklar und somit in vitro-Klasse-Schalter Assays bieten eine zuverlässige Methode zur Prüfung dieser Prozesse in verschiedenen Mausmodellen. Diese Assays wurden bereits im Zusammenhang mit der Gen-Mangel mit Knockout-Mäusen 3 verwendet. Darüber hinaus können in vitro Schalten von B-Zellen durch virale Transduktion vorangestellt werden, um die Genexpression durch RNA-Zuschlag oder Transgenexpression 4-6. Die Daten aus dieser Art von Experimenten haben unser Verständnis der AID-Aktivität, die Auflösung der CSR-Reaktion und Antikörper-vermittelte Immunität in der Maus beeinflusst.

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

Nach Antigen Exposition unterziehen Subpopulationen von aktivierten B-Zellen ein Verfahren, wie class switch Rekombination (CSR) bekannt, dass Antikörper-Isotypen mit unterschiedlichen Effektorfunktionen zu produzieren. Das Protokoll in diesem Bericht beschriebenen erklärt, wie CSR induziert und analysiert werden können<em&gt; In-vitro-</em&gt; Für die Zwecke des Studiums B-Zell-Funktion.

Induktion und Beurteilung der Class Schalter Rekombination in Purified Murine B-Zellen

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

18.1K Aufrufe.  University of Toronto. Nach Antigen Exposition unterziehen Subpopulationen von aktivierten B-Zellen ein Verfahren, wie class switch Rekombination (CSR) bekannt, dass Antikörper-Isotypen mit unterschiedlichen Effektorfunktionen zu produzieren. Das Protokoll in diesem Bericht beschriebenen erklärt, wie CSR induziert und analysiert werden können In-vitro- Für die Zwecke des Studiums B-Zell-Funktion.

Serie: 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.

Mausmodell der CD40-Aktivierung von B-Zellen

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

Forschung

Immunologie und Infektion

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.

Rekombinanten retroviralen Produktion und Infektion von B-Zellen

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 ...

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.

Identifizierung von DNA-Mutationen in hämatopoetischen Stammzellen Gereinigtes / Vorläuferzellen

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.

Der Nachweis von IgE-Wahre exprimierenden Maus B Lineage Cells

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.

Die Induktion von Murine Darmentzündungen durch adoptiven Transfer von Effektor CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Hoch</sup&gt; T-Zellen in immundefizienten Mäusen

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.

Beurteilung der immunmodulatorischen Eigenschaften von humanen mesenchymalen Stammzellen (MSCs)

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.

Ein Mausmodell der Gruppe B<em&gt; Streptococcus</em&gt; Vaginal Colonization

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

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Durchflusszytometrische Analyse von partikelgebundenen Bet v 1 Allergens in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

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.

Genomweite Analyse von HDAC-Inhibitor-vermittelten Modulation der Micro-RNAs und mRNAs in B Zellen induziert unterziehen Class-Schalter DNA-Rekombination und Plasma-Zell-Differenzierung

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 und In Vivo Beurteilung der T, B und myeloische Zellen Suppressive Aktivität und humorale Reaktionen von Transplantationspatienten

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