Name: detection of true ige-expressing mouse b lineage cells
Uploaded: 2014-12-01T14:00:00.000+00:00
Duration: 9 min 40 s
Description: Watch this Scientific Journal Video about Detection of True IgE-expressing Mouse B Lineage Cells at JoVE.com

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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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><tbody><tr><td>Anti-Mouse IgE FITC</td><td>BD Pharmingen</td><td>553415</td><td>clone R35-72</td></tr><tr><td>Rat Anti-Mouse IgG1 PE</td><td>BD Pharmingen</td><td>550083</td><td>clone A85-1</td></tr><tr><td>Methanol</td><td>BDH</td><td>BDH1135-4LP</td><td>Keep at -20 &amp;deg;C</td></tr><tr><td>Falcon Cell Strainer 40 &amp;micro;m Nylon</td><td>Corning</td><td>352340</td><td></td></tr><tr><td>Falcon Cell Strainer 70 &amp;micro;m Nylon</td><td>Corning</td><td>352350</td><td></td></tr><tr><td>Anti-Hu/Mo CD45R(B220) PerCP-Cy5.5</td><td>eBioscience</td><td>45-4052-82</td><td>clone RA3-6B2</td></tr><tr><td>anti-Mouse/Rat CD40</td><td>eBioscience</td><td>16-0402-85</td><td>clone HM40-3</td></tr><tr><td>RPMI Medium 1640</td><td>Gibco</td><td>11875-093</td><td></td></tr><tr><td>HEPES (1M)</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>MEM-NEAA (100x)</td><td>Gibco</td><td>11140-050</td><td></td></tr><tr><td>Phosphate Buffered Saline (10x)</td><td>Lifetechnologies (Corning)</td><td>46-013-CM</td><td></td></tr><tr><td>MACS CD45R (B220) microbeads&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-049-501</td><td></td></tr><tr><td>MACS purification column</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>IL-4</td><td>PeproTech</td><td>214-14</td><td>Reconstitute in water or 0.1% BSA</td></tr><tr><td>Formalin Solution, Neutral Buffered, 10%</td><td>Sigma Aldrich</td><td>HT501128-4L</td><td></td></tr><tr><td>Trypsin-EDTA Solution (10x)</td><td>Sigma Aldrich</td><td>T4174-100 ml</td><td>5.0 g Trypsin, 2.0 g EDTA&amp;#8226;4 Na per Liter of 0.9% NaCl</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma Aldrich</td><td>P0781-100 ml</td><td>10,000 U Penicillin; 10 mg/ml Streptomycin (100x)</td></tr><tr><td>L-Glutamine 200 mM</td><td>Sigma Aldrich</td><td>G7513</td><td></td></tr><tr><td>2-mercaptoethanol (99%)</td><td>Sigma Aldrich</td><td>M6250-100 ml</td><td></td></tr><tr><td>Red cell lysis buffer</td><td>Sigma Aldrich</td><td>R7757</td><td></td></tr><tr><td>Rat Anti-Mouse IgM RPE/Cy7</td><td>SouthernBiotech</td><td>1140-17</td><td>clone 1B4B1</td></tr><tr><td>HyClone Fetal Calf Serum</td><td>Thermo</td><td>SH30910.03</td><td></td></tr><tr><td>B cell Stimulation media</td><td></td><td></td><td>B cell stimulation media consists of RPMI with fetal calf serum (15%), 20 mM HEPES, 1x MEM-NEAA, 2.0 mM L-glutamine,&amp;nbsp; 1x Penicillin-Streptomycin (Penicillin: 100 U/ml, Streptomycin: 100 &amp;micro;g/ml), Beta-mercaptoethanol (7 &amp;micro;L/L), IL-4 (20 ng/ml), and anti-CD40 (1 &amp;micro;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

10.9K 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

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 ...

Forschung

Immunologie und Infektion

Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable cytokine-secreting cells. In order to label IL-17-secreting cells, a single cell suspension of mouse splenocytes is prepared and stimulated at 37&deg;C with PMA/ionomycin to induce cytokine secretion. To stop secretion cells are then placed on ice and are exposed to the IL-17 Catch Reagent   a bi-specific antibody that binds to CD45 on the cell surface of leukocytes and to IL-17 as it is secreted and caught near the cell surface. Secretion is then re-started by increasing the temperature to 37&deg;C and IL-17 is trapped by the Catch Reagent. Secretion is then stopped again, by placing cells on ice. To detect the trapped IL-17, cells are incubated with a second IL-17-specific antibody conjugated to biotin and an Anti-Biotin-PE antibody. Cells can now be directly analyzed by flow cytometry or prepared for isolation and enrichment by subsequent labeling with Anti-PE conjugated MicroBeads.

This procedure describes the detection and isolation of mouse TH17 leukocytes that actively secrete IL-17 upon stimulation.

Detektion und Isolation von lebensfähigen Maus IL-17-sezernierenden T-Zellen

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable ...

Biologie

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 ...

The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible animal models mirror common components of human disease, including the bm12 transfer model. Upon transfer of bm12 splenocytes or purified CD4 T cells, C57BL/6 mice rapidly develop large frequencies of T follicular helper cells (Tfh), germinal center (GC) B cells, and plasma cells followed by high levels of circulating anti-nuclear antibodies. Since this model utilizes mice on a pure C57BL/6 background, researchers can quickly and easily study disease progression in transgenic or knockout mouse strains in a relatively short period of time. Here we describe protocols for the induction of the model and the quantitation Tfh, GC B cells, and plasma cells by multi-color flow cytometry. Importantly, these protocols can also be used to characterize disease in most mouse models of SLE and identify Tfh, GC B cells, and plasma cells in other disease models.

The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice

The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.

Das BM12 Inducible Modell des systemischen Lupus erythematodes (SLE) in C57BL / 6-Mäuse

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible ...

Medizin

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. 
The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.

A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly &#x2013;harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.

Identifikation und Analyse von Maus Erythroid Vorläufern mit dem CD71/TER119 Durchflusszytometrische Assay

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

Charakterisierung der Thymus-abhängig und Thymus-unabhängige Immunglobulin Isotype Antworten bei Mäusen mit Enzym-linked Immunosorbentprobe Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

Here we describe a method for retroviral overexpression and adoptive transfer of murine B-1a cells to examine in vivo B-1a cell migration and localization. This protocol can be extended for diverse downstream functional assays including quantification of donor B-1a cell localization or analysis of donor cell-derived secreted factors post-adoptive transfer.

Retrovirale Überexpression von CXCR4 auf murine B-1a-Zellen und Adoptivtransfer für gezielte B-1a-Zellmigration zur Knochenmark- und IgM-Produktion

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide ...

Flow Cytometric Characterization of Murine B Cell Development

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

Durchflusszytometrische Charakterisierung der Entwicklung von Murinen B-Zellen

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells ...

Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while suppressing unwanted reactions to environmental antigens. Lungs host several populations of innate and adaptive immune cells that provide immune surveillance but also mediate protective immune responses. These cells, which keep the healthy pulmonary immune system in balance, also participate in several pathological conditions such as asthma, infections, autoimmune diseases, and cancer. Selective expression of surface and intracellular proteins provides unique immunophenotypic properties to the immune cells of the lung. Consequently, flow cytometry has an instrumental role in the identification of such cell populations during steady-state and pathological conditions. This paper presents a protocol that describes a consistent and reproducible method to identify the immune cells that reside in the lungs of healthy mice under steady-state conditions. However, this protocol can also be used to identify changes in these cell populations in various disease models to help identify disease-specific changes in the lung immune landscape.

In this study, we present an effective and reproducible protocol to isolate the immune populations of the murine respiratory system. We also provide a method for the identification of all innate and adaptive immune cells that reside in the lungs of healthy mice, using a 9-color-based flow cytometry panel.

Durchflusszytometrische Analyse zur Identifizierung der angeborenen und adaptiven Immunzellen der murinen Lunge

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while ...

Isolation of Group 2 Innate Lymphoid Cells from Mouse Nasal Mucosa to Detect the Expression of CD226

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various pathological processes, including anti-helminth immunity, tissue repair, thermogenesis, and autoimmune diseases such as asthma and allergic rhinitis (AR). ILC2s permanently reside in peripheral tissues such as the skin, gut, lungs, and nasal cavity; however, there is limited information about their exact functions in nasal mucosal immunity. CD226 is an activating costimulatory molecule, mainly expressed on natural killer (NK) cells, T cells, and inflammatory monocytes. However, whether ILC2s express CD226 or play a role in the pathogenesis of ILC2s-related diseases remains unknown. Here, we established a method to isolate and identify ILC2s from the nasal mucosa and detected CD226 expression on ILC2s obtained from healthy and AR mice. Herein, we describe this protocol for the isolation and identification of ILC2s from mouse nasal mucosa, which will help explore the internal pathological mechanism of immunological disorders in nasal mucosal diseases.

Group 2 innate lymphoid cells (ILC2s), implicated in type 2 inflammation, mainly participate in response to helminth infection, allergic diseases, metabolic homeostasis, and tissue repair. In this study, a procedure to isolate ILC2s from murine nasal mucosa and detect the expression of CD226 is demonstrated.

Isolierung von angeborenen lymphatischen Zellen der Gruppe 2 aus der Nasenschleimhaut der Maus zum Nachweis der Expression von CD226

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various ...