DRW wird durch NIH Zuschüsse AI089972 und AI113217 und durch die Mukosaimmunologie Studies-Team unterstützt, und hat einen Career Award für medizinische Wissenschaftler der Burroughs Wellcome Fund.

HINWEIS: Alle hier beschriebenen Experimente wurden in Übereinstimmung mit den Institutional Animal Care und Verwenden Committee (IACUC) Richtlinien und von Tierforschung Kinderkrankenhaus (ARCH), Boston, Mass zugelassen. 1. Vorbereitung der Reagenzien <ol><li> 1,000x IL-4 Stock: Verdünnen IL-4 Cytokins an 20 ug / ml in H 2 O oder 0,1% Rinderserumalbumin (BSA). Dispense in 200 ul Aliquots in 1,5 ml Mikrozentrifugenröhrchen und bei -20 ° C. </li><li> 1,000x anti-CD40: Beziehen des Herstellers bei 1,0 mg / ml, Lagerung bei 4 ° C. </li><li> B-Zell-Stimulation Mittel: Auf 425 ml RPMI-1640-Medium, fügen Sie 75 ml frisch aufgetauten fötalem Kälberserum (FCS), 10 ml 1,0 M HEPES-Puffer, 5 ml L-Glutamin Lager, 5 ml Penicillin: Streptomycin Lager, 5 ml Minimum Essential Medium nicht essentiellen Aminosäuren (MEM-NEAA) und 3,5 ul 2-Mercaptoethanol. Lagern Sie Druckmaterial bei 4 ° C für bis zu einer Woche. Hinzufügen IL-4 (20 ng / ml) und Anti-CD40 (1.0 ug / ml) unmittelbar vor der Anwendungnur auf das Volumen des benötigten Medien. </li><li> 2% FCS-FACS-Puffer: 10 ml FCS bis 490 ml 1x PBS. Halten bei 4 ° C. </li><li> 2x Trypsin-EDTA-Lösung: 5 ml der 10fach Trypsin-EDTA-Stammlösung (5,0 g Trypsin, 2,0 g EDTA pro Liter) in 20 ml 1 × PBS. Speicher Trypsin-EDTA in 2x Arbeits Aliquots bei 4 ° C. Ermöglichen, sich auf Raumtemperatur gebracht werden. </li><li> Fötalem Kälberserum: Halten aufgetauten FCS bei 4 ° C für bis zu zwei Wochen. Zeigen auf Eis 15 min vor der zytoplasmatischen Färbungsverfahren. </li><li> Neutral-Formalin: Eine 10% ige Formalin-Lösung (3,7% Paraformaldehyd). Formalin ist giftig und daher behandeln es unter einem Laborabzug beim Tragen geeigneter PSA. Shop Formalin bei Raumtemperatur in einer chemischen Speicherschrank. </li><li> Methanol: Shop absolutem Methanol (100%) bei -20 ° C, bis die Zellen bereit sind, durchlässig gemacht werden. Beachten Sie, dass Methanol ist brennbar und sollte in einem angemessenen Gefrierschrank gelagert werden. </li></ol> 2.Reinigung und Aktivierung von B-Zellen <ol><li> Von einem frisch geerntet Mäusemilz, bereiten eine Einzelzellsuspension durch leichtes Eindrücken der Orgel durch eine 70 um Zellsieb mit dem Kolben eines 3-ml-Spritze. In ein 15 ml konisches Zentrifugenröhrchen sammeln Zellen in 10 ml 1x PBS und Zentrifugation bei 300 × g für 5 min bei 4 ° C. </li><li> Dekantieren Sie den Überstand und Pellet in 1 ml Rotes Blutkörperchen Lysepuffer für 5 min. Neutralisieren mit 13,5 ml 1 × PBS und Zentrifuge bei 300 xg für 5 min. </li><li> (Optional) magnetisches Trennen der B-Zellen mit anti-CD45R (B220) -markierten Kügelchen nach der MACS Reinigungsprotokoll gemäß den Anweisungen des Herstellers. </li><li> Stimulieren gereinigte B-Zellen mit 1,0 × 10 6 Zellen / ml in erwärmt B-Zell-Stimulation Medien, die IL-4 (20 ng / ml) und Anti-CD40 (1.0 ug / ml) für 5 Tage bei 37 ° C. Beginnen Sie mit 8 ml der Kultur in 2 Vertiefungen einer 6-Well-Kulturplatte (je 4 ml) aufgeteilt. </li><li> Überprüfen cultuWieder täglich mit 1,0 x 10 6 Zellen / ml zu erhalten. Die Konzentration ist anzupassen durch Aufteilen in andere Vertiefungen und mit B-Zell-Stimulationsmittel mit frischem IL-4 und anti-CD40 Verdünnen der Kultur. </li></ol> 3. Trypsinisierung, Fixierung und Permeabilisierung <ol><li> Trypsinierung <ol><li> Sammeln von bis zu 1,0 x 10 7 kultivierten Zellen in einem 15 ml konischen Röhrchen, Zentrifuge bei 300 × g für 5 Minuten und dekantiert den Überstand. </li><li> Waschen mit 10 ml PBS, um die Rest Proteins aus der Zelle Mappen Medien zu entfernen. Wiederholen Sie den Zentrifugationsschritt und dekantiert die überstehende. </li><li> Zellpellet in dem Restvolumen nach dem Dekantieren (etwa 100 ul). </li><li> 800 ul der Raumtemperatur 0,1% (2x) Trypsin-EDTA sorgfältig in den Zellen. Verzichtet werden langsam in einem Winkel von 45 ° an der Seite des konischen Rohrs. </li><li> Die Tube und vorsichtig durch Kippen des Rohrs, so dass die Flüssigkeit entlang der Länge des Rohres 5-6 Zeit abläufts. Eine fadenziehende, weiße Niederschlag in der Lösung zu bilden. Begrenzen Sie die Zeit, um Trypsinisierung unter min. </li><li> Quenche die Trypsinisierung Reaktion mit 1 ml kaltem FCS. Verdünne das FCS sofort durch Zugabe von 10 ml kaltem PBS gewaschen. </li><li> Zentrifuge bei 300 × g für 5 min bei 4 ° C, dann dekantiert und die Röhrchen zurück zu Eis. </li></ol></li><li> Fixierung <ol><li> Resuspendieren der Zellen in dem Restvolumen nach dem Dekantieren. Falls erforderlich, bringen Volumen von bis zu 100 &amp; mgr; l mit kaltem PBS. </li><li> In einer Abzugshaube, fügen Sie 800 ul von 10% neutral gepuffertem Formalin-Lösung zu den Zellen und pipettieren auf und ab. Das Rohr wird durch die Zugabe von Formalin zu erwärmen. Bei 37 ° C für 10 min. </li></ol></li><li> Permeabilisierung <ol><li> Richten Sie eine Tisch Vortex-Mischer in einem leichtem Schütteln Geschwindigkeit. </li><li> Zeichnen 9 ml kaltem (-20 ° C) Methanol in einen 10 ml serologische Pipette. </li><li> Halten Sie das Röhrchen mit den Zellen über die Vortex-Mischer und sanft starten Shaking die Zellen. </li><li> Kaltes Methanol zu dem Rohr so ​​dass es auf den Zellen tropfenweise fällt, während das Rohr noch Schütteln, so dass das Methanol kontinuierlich gemischt. Nach den ersten 2 ml Methanol kann die Abgabegeschwindigkeit zu einem langsamen Strom erhöht werden. </li><li> Lassen Sie den Schlauch sitzen auf Eis für 30 min, um die Permeabilisierung abzuschließen. Alternativ können die Zellen bei -20 ° C für bis zu einem Monat gelagert werden. </li></ol></li></ol> 4. Fluoreszenzfärbung für IgG1 und IgE Class Switching <ol><li> Centrifuge Probe bei 300 xg für 5 min und dekantiert die Methanollösung. Ein weißer Niederschlag kann mit den Zellen zu pelletieren. </li><li> 10 ml Raumtemperatur 1x PBS und Zentrifuge bei 300 xg für 5 min, um Zellen zu waschen. Wiederholen Sie einmal. Hinweis: Das Waschen des Pellets mit Raumtemperatur PBS löst zusätzliche Niederschlag, der nach dem Schritt 4.1 bilden kann. </li><li> Führen Sie eine weitere Waschung mit 10 ml 2% FCS-FACS-Puffer. </li><li> Verteilen Proben in eine 96-Well-Rundbodenplatteund Zentrifuge bei 300 xg für 5 min. Dekantieren und stellte den Teller auf dem Eis. </li><li> Stain in 60 ul 2% FCS-FACS-Puffer pro Vertiefung mit folgenden Antikörperverdünnungen: 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> Nach 5 min Färbung, waschen der Platte mit 150 ul 2% FCS-FACS-Puffer und Zentrifuge 5 min bei 300 x g. </li><li> Resuspendieren in 400 ul 2% FCS-FACS-Puffer und Pipette durch ein 40 um Nylon Zellsieb in ein 5 ml FACS-Röhrchen. </li></ol> 5. Verwenden Sie die folgenden Gating-Strategie: <ol><li> Isolieren CD45R (B220) positive Zellen aus dem Spreng Lymphozyten-Population auf der FSC / SSC Plot (Abbildung 1) befindet. </li><li> Von der B220-positiven Zellen, verwenden Sie die IgE und IgG1 Grundstück, um die entsprechenden Populationen zu offenbaren. </li></ol>

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The authors have no disclosures.

Stimulation von Maus-Milz-B-Zellen in Kultur mit Anti-CD40 und IL-4 wird T-Helfer Typ 2 (TH 2) Wechselwirkungen, ermutigend Klassenumschaltzeit IgG1 und IgE 5 simulieren. B-Zellen können für CSR im Rahmen der Gesamt Splenozyten 12 oder als gereinigte Milz-B-Zellen, 11 aktiviert werden. Wie in dem Protokoll (Schritt 2.3) angemerkt, ist B-Zell-Anreicherung optional, und liegt im Ermessen des Experimentators, ob es von Vorteil, zu ermitteln. Positive magnetische Trennung von B-...

Während der schweren Immunglobulinkette (IgH) Klassenwechsel-Rekombination (CSR) in Mäusen und Menschen, μ die IgH Exons der konstanten Region (C &amp; mgr;) gelöscht und durch eine von mehreren Sätzen von nachgeschalteten Exons der konstanten Region (C H s) (z Cy ersetzt, C &amp; egr; und C &amp; alpha;), was zu einem Wechsel von der Herstellung von IgM auf die Herstellung von anderen Ig-Klassen (beispielsweise IgG, IgE oder IgA). CSR tritt innerhalb Schalter (S) Regionen, die 1-10 kb-Sequenzen, die 5 &#39;an jeden Satz von C H s 1 sind. Die Aktivierungs-induzierte Cytidindeaminase (AID) Enzym initiiert CSR über Cytidin Desaminierung Aktivität. IgE ist ein wichtiger Mediator der allergischen Erkrankung 2 und ein besseres Verständnis dafür, wie IgE produziert und reguliert kann Türen zu neuen Therapieansätzen zu atopischen Erkrankung zu öffnen. In Mäusen und Menschen, ist die IgE streng reguliert Ig Isotyp. IgE wird normalerweise in Mengen Tausende von tim erkanntes weniger als andere IgH Isotypen 3, kann jedoch stark bei Krankheitszuständen 4 erhöht werden. Jedoch CSR an IgE ist nicht vollständig verstanden. In vitro-Aktivierung von B-Zellen mit IL-4 in Kombination mit entweder Anti-CD40 oder LPS induziert CSR sowohl IgG1 und IgE 5. Aktivierte B-Zellen exprimieren den niedrigen affinen IgE-Rezeptors FcεRII / CD23 2,6, die die lösliche IgE in Kultur nach CSR abgesondert bindet. Deshalb bei der Analyse mit Durchflusszytometrie, B-Zellen mit rezeptorgebundenen IgE-Färbung ähnlich wie Zellen endogen exprimierenden IgE 7b. Obwohl bekannt ist, dass die Maus-B-Zellen das geringe Affinität IgG-Rezeptor FcγRIIB1 (CD32) 8, in unserer Erfahrung ist es offenbar nicht mit der Messung der Klassenwechsel zu IgG1 stören. Allerdings, wenn die Messung IgE Schalt nach B-Zell-Aktivierung in der Kultur, nicht spezifische oberflächengebundene IgE kann die Analyse verschleiern. Mit gemeinsamen Färbeverfahren, Fleck nicht IgE-exprimierenden Zellen positiv für IgE. Hier skizziert ist eine Strategie, die verwendet worden ist, um CSR-Tests von Mäusen 9 durchführen und erkennen wahre IgE-exprimierenden Zellen, B - 11. Behandlung von aktivierten B-Zellen mit Trypsin, einem gemeinsamen Labor-Reagens, um Protein zu verdauen, entfernt sowohl cytophylic und Membran-gebundenen Oberfläche IgE. Nachfolgende Permeabilisierung und Färbung für zytoplasmatische IgE zeigt somit die wahren IgE-produzierenden Zellen.

<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-Lymphozyten-Immunglobulin schwere Kette (IgH) Klassenwechsel-Rekombination (CSR) ist ein Verfahren, bei dem zunächst ausgedrückt IgM schaltet auf andere IgH Isotypen, wie IgA, IgE und IgG. Messung der IgH CSR in vitro ist ein Schlüsselverfahren für die Untersuchung einer Reihe von biologischen Prozessen, die von DNA-Rekombination und Reparatur, um Aspekte der molekularen und zellulären Immunologie. In vitro CSR Assay das durchflusszytometrische Meßfläche Ig-Expression auf aktivierten B-Zellen. Während Messung des IgA- und IgG-Subklassen ist einfach, ist die Messung von IgE durch dieses Verfahren problematisch aufgrund löslichen IgE-Bindung an FcεRII / CD23, das auf der Oberfläche von aktivierten B-Zellen. Hier beschreiben wir ein einzigartiges Verfahren für die genaue Messung von IgE-produzierende Maus-B-Zellen, die CSR in Kultur erfahren haben. Das Verfahren basiert auf Trypsin-vermittelten Spaltung von IgE-CD23-Komplexen auf der Zelloberfläche gehalten und erlaubt die Detektion von IgE-produzierenden B-Linie Zellen durch cytoplasmic Färbung. Dieses Verfahren bietet eine komfortable Lösung für die durchflusszytometrische Analyse von CSR an IgE.

Dieses Verfahren wurde erfolgreich durchgeführt, um CSR an IgE in Maus-B-Zellen zu untersuchen. Um die Effizienz bei der Messung CSR demonstrieren angeregt wir Maus Milz-B-Zellen mit anti-CD40 und IL-4, wie zuvor beschrieben 11. Nach fünf Tagen der Stimulation wurden die Zellen gesammelt und mit dem vorstehend beschriebenen und mit fluoreszierend markierten IgM, IgG1, IgE und B220 (CD45R) Antikörpern gefärbt Protokoll verarbeitet. An Strahl Lymphozyten (1), eine klare Population von IgE +...

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.

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 ...

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

Research

JoVE Journal

Immunology and Infection

10.8K Aufrufe.  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.

Serie: 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.

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

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.

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 ...

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 ...

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.

Schnelle Analyse von Chromosomenaberrationen in Maus-B-Lymphozyten durch 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.

Erzeugung von rekombinanten humanen IgG monoklonalen Antikörpern aus Immortalisierte Sortiert B Cells

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.

Schnelle molekulare Erkennung und Differenzierung von Influenza-Viren A und 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.

Detektion und Anreicherung von Rare Antigen-spezifische B-Zellen für die Analyse von Phänotypen und Funktion

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.

Erkennung von Inflammasome Aktivierung und Pyroptotic Zelltod in murinen Makrophagen Knochenmark abgeleitet

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.

Identifizierung und Isolierung von Oligopotent und Lineage-committed myeloischen Vorläuferzellen aus Maus Knochenmark

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.

Erkennung von niedrigen Kopie Nummer integrierte virale DNA gebildet durch In-vitro- Hepatitis B-Infektion

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