Name: an in vivo mouse model to measure na&#239;ve cd4 t cell activation, proliferation and th1 differentiation induced by bone marrow-derived dendritic cells
Uploaded: 2018-08-22T15:00:00
Description: Watch this Scientific Journal Video about An In Vivo Mouse Model to Measure NaÃ¯ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells at JoVE

Wir danken Dr. Simon Bartlett für englische Bearbeitung. Diese Studie wurde unterstützt durch Zuschüsse vom Instituto de Salud Carlos III (ISCIII) (PI14/00526; PI17/01395; CP11/00145; CPII16/00022), Miguel Servet Programm und Fundación Ramón Areces; mit Kofinanzierung von Fondo Europeo de Desarrollo Regional (FEDER). Das CNIC wird unterstützt durch das Ministerium für Wirtschaft, Industrie und Wettbewerbsfähigkeit (MEIC) und der Stiftung Pro CNIC und ist ein Severo Ochoa Center of Excellence (SEV-2015-0505). RTF ist gegründet von Fundación Ramón Areces und CNIC, VZG durch ISCIII, BHF von Instituto de Investigación Sanitaria Krankenhaus 12 de Octubre (imas12) und JMG-G von den ISCIII Miguel Servet Programm und imas12.

Experimentelle Verfahren wurden genehmigt durch die Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC) und der Comunidad Autónoma de Madrid nach spanischen und europäischen Richtlinien. Mäuse wurden in spezifischen Erreger frei (SPF) Bedingungen gezüchtet und durch Kohlendioxid (CO2) Inhalation eingeschläfert wurden.
1. Isolierung von Knochenmarkzellen Maus aus Gebeinen und Oberschenkelknochen
Hinweis: Der C57BL/6 Congenic...

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J., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effector+and+memory+T-cell+differentiation:+implications+for+vaccine+development.">Effector and memory T-cell differentiation: implications for vaccine development.</a> Nature Reviews Immunology. 2 (4), 251-262 (2002).</li><li>Iborra, S., Gonzalez-Granado, J. 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Dieses Protokoll ermöglicht die Charakterisierung der Kapazität der BM-abgeleitete DCs die Aktivierung, Proliferation und Differenzierung von naiven CD4-T-Zellen zu modulieren. Darüber hinaus es auch lässt sich beurteilen, die Anfälligkeit der CD4-T-Zellen zur Modulation von DCs BM abgeleitet. Mit diesem Protokoll kann Änderungen an diesen Veranstaltungen gemessenen in Vivo.
Je nach der Hypothese untersucht können mehrere Kombinationen von T-Zellen und DCs verwendet werden. Zum...

CD4-T-Zellen und antigenpräsentierende Zellen (APCs) wie DCs sind erforderliche Mediatoren der Immunität gegen mikrobiellen Krankheitserregern1,2. In den peripheren lymphatischen Organen werden CD4-T-Zellen bei der Anerkennung der spezifischen Antigene präsentiert von APCs3,4,5aktiviert. Aktivierte CD4-T-Zellen vermehren sich und differenzieren in verschiedene spezifische Th Effektorzellen, die notwendig sind für die Entwicklung eines korrekten adaptiven Immunantwort6,7. Kontrolle dieser Prozesse ist entscheidend für die Herstellung einer angemessenen adaptive Verteidigung, die den Erreger abtötet ohne schädliche Gewebe Schaden8zu produzieren. Th-Zellen werden nach den Ausdruck oder die Produktion von Oberflächenmoleküle, Transkriptionsfaktoren und Effektor Zytokine definiert und wesentliche und präzise Funktionen in Reaktion auf Krankheitserreger1. Zellen der Th1-Zelle Teilmenge Transkriptionsfaktors T-Bet und das Zytokin Interferon-γ (IFNγ) Ausdrücken und in der Wirt Verteidigung gegen intrazelluläre Erreger1teilnehmen. Quantifizierung der naiven CD4-T-Zell-Aktivierung, Proliferation und Differenzierung der Th1 ist ein nützliches Mittel zur Beurteilung der T Zellen Rolle bei einer Immunantwort.
Dieses Protokoll ermöglicht in Vivo Analyse der Leistungsfähigkeit der in Vitro -generiert BM abgeleitet DCs die Aktivierung, Verbreitung und Th1 Differenzierung der naiven CD4-T-Zellen zu modulieren. Das Protokoll dient auch dazu, die Fähigkeit der naiven CD4-T-Zellen aktiviert, induzierte zu vermehren geprüft und Th1 differenziert (Abbildung 1). Dieses vielseitige Protokoll umgeht die Unfähigkeit, speziell zu manipulieren oder die Studium Zellpopulation in rein in Vivo Protokolle auswählen. Die Auswirkungen von unterschiedlichen Molekülen und Behandlungen auf Domänencontrollern können mit BM von genetisch veränderten Mäusen5 oder durch Behandlung oder genetisch manipulieren isoliert BM Zellen9untersucht werden. T-Zell-Antworten können ebenso erkundet werden, durch den Erwerb von T-Zellen für Adoptiv-Transfer aus unterschiedlichen Quellen oder nach mehreren Manipulationen3,8,10.
Die wichtigsten Vorteile dieses Protokolls sind von zweierlei Art. T-Zell-Aktivierung, Proliferation und Differenzierung der Th1 sind mit einem Flow Cytometry Ansatz analysiert; und dies wird kombiniert mit in Vivo Studien, somit Abwendung Veränderungen, die in Vitro auftreten können sowie Zelltypen und andere Faktoren, die nur in intakten Organe11gefunden.
Die Verwendung von lebenswichtigen Farbstoffen ist eine weit verbreitete Technik, Zellproliferation und vermeiden den Gebrauch von Radioaktivität zu verfolgen. Die Messung der Verbreitung mit dieser Reagenzien basiert auf Farbstoff Verdünnung nach der Zellteilung. Darüber hinaus diese Farbstoffe können bei mehreren Wellenlängen erkannt werden und sind leicht durch Durchflusszytometrie in Kombination mit mehreren fluoreszierende Antikörper oder Marker analysiert. Wir markieren die Nützlichkeit dieses Protokoll zeigt, wie die T-Zellaktivierung, Proliferation und Differenzierung der Th1 durch Durchflusszytometrie analysiert werden können.

<table>
	<tbody>
		<tr>
			<td>Ethanol</td>
			<td>VWR Chemicals</td>
			<td>20,824,365</td>
			<td>5 L</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools (F.S.T.)</td>
			<td>14001-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools (F.S.T.)</td>
			<td>11000-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Fine Science Tools (F.S.T.)</td>
			<td>11253-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Fine Science Tools (F.S.T.)</td>
			<td>10020-00</td>
			<td>Box of 100 blades</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>SIGMA</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>LONZA</td>
			<td>DE17-602E</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute medium (RPMI)</td>
			<td>GIBCO</td>
			<td>21875-034</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Petri dishes</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>25-gauge needle</td>
			<td>BD Microlance 3</td>
			<td>300600</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>Novico</td>
			<td>N15663</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conical tubes</td>
			<td>Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conical tubes</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m nylon web filter</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood lysis buffer</td>
			<td>SIGMA</td>
			<td>R7757</td>
			<td>100 Ml</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>SIGMA</td>
			<td>ED2SS-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>SIGMA</td>
			<td>A7906</td>
			<td>100 g</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>SIGMA</td>
			<td>302643-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture-plates</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td>Cambrex</td>
			<td>BE17-737E</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta-mercaptoethanol</td>
			<td>Merck</td>
			<td>8-05740-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>LONZA</td>
			<td>BE13-115E</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>LONZA</td>
			<td>BE17-605E</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant murine Granulocyte Macrophage colony-stimulating factor (GM-CSF)</td>
			<td>Prepotech</td>
			<td>315-03</td>
			<td></td>
		</tr>
		<tr>
			<td>lipopolysaccharide (LPS)</td>
			<td>SIGMA-ALDRICH</td>
			<td>L2018</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well-plate</td>
			<td>Costar</td>
			<td>3799</td>
			<td></td>
		</tr>
		<tr>
			<td>v450 anti-mouse CD11b antibody</td>
			<td>BD Biosciences</td>
			<td>560455</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-mouse CD64 antibody</td>
			<td>BioLegend</td>
			<td>139303</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-mouse CD115 antibody</td>
			<td>BioLegend</td>
			<td>135505</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD11c antibody</td>
			<td>BioLegend</td>
			<td>117305</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse MHCII antibody</td>
			<td>BioLegend</td>
			<td>125507</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD86 antibody</td>
			<td>BioLegend</td>
			<td>105011</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD80 antibody</td>
			<td>BioLegend</td>
			<td>104713</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometry tubes</td>
			<td>Zelian</td>
			<td>300800-1</td>
			<td>PS 12X75 5mL</td>
		</tr>
		<tr>
			<td>OTII Ovoalbumine peptide</td>
			<td>InvivoGen</td>
			<td>323-339</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD8&#945; antibody</td>
			<td>Tonbo Biosciences</td>
			<td>30-0081-U500</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated IgM antibody</td>
			<td>BioLegend</td>
			<td>406503</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated B220 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>30-0452-U500</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD19 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>30-0193-U500</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated MHCII (I-Ab) antibody</td>
			<td>BioLegend</td>
			<td>115302</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD11b antibody</td>
			<td>Tonbo Biosciences</td>
			<td>30-0112-U500</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD11c antibody</td>
			<td>BioLegend</td>
			<td>117303</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD44 antibody</td>
			<td>BioLegend</td>
			<td>103003</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD25 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>30-0251-U100</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated DX5 antibody</td>
			<td>BioLegend</td>
			<td>108903</td>
			<td></td>
		</tr>
		<tr>
			<td>streptavidin-coated magnetic microbeads</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-048-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic cell separator</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td>QuadroMACS Separator</td>
		</tr>
		<tr>
			<td>Separation columns</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-mouse CD4 antibody</td>
			<td>BioLegend</td>
			<td>100408</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD3 antibody</td>
			<td>BioLegend</td>
			<td>100235</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD8 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>35-0081-U025</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Violet Tracer</td>
			<td>Thermofisher</td>
			<td>C34557</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD25 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>20-0251-U100</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa647 anti-mouse CD69 antibody</td>
			<td>BioLegend</td>
			<td>104518</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCPCY5.5 anti-mouse CD45.1 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>65-0453</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD45.1 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>20-0453</td>
			<td></td>
		</tr>
		<tr>
			<td>PECY7 anti-mouse CD45.1 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>60-0453</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD45.2 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>35-0454</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>SIGMA-ALDRICH</td>
			<td>I0634</td>
			<td></td>
		</tr>
		<tr>
			<td>Phorbol 12 Myristate 13 Acetate (PMA)</td>
			<td>SIGMA</td>
			<td>P8139</td>
			<td></td>
		</tr>
		<tr>
			<td>Brefeldin A (BD GolgiPlug)</td>
			<td>BD</td>
			<td>555029</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Millipore</td>
			<td>8-18715-02100</td>
			<td></td>
		</tr>
		<tr>
			<td>Intracellular permeabilization buffer</td>
			<td>eBioscience</td>
			<td>00-8333</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse IFNg antibody</td>
			<td>Tonbo Biosciences</td>
			<td>20-7311-U100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc-block (anti-mouse CD16/CD32)</td>
			<td>Tonbo Biosciences</td>
			<td>70-0161-U100</td>
			<td></td>
		</tr>
		<tr>
			<td>B6.SJL CD45.1 mice</td>
			<td>The Jackson Laboratory</td>
			<td>002014</td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSRFortessa&#8482; Cell Analyzer</td>
			<td>BD Biosciences</td>
			<td>649225</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI Solution</td>
			<td>Thermofisher</td>
			<td>62248</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vivo mouse model to measure na&#239;ve cd4 t cell activation, proliferation and th1 differentiation induced by bone marrow-derived dendritic cells

Quantifizierung der naiven CD4-T-Zell-Aktivierung, Proliferation und Differenzierung zu T-Helfer 1 (Th1) Zellen ist eine nützliche Methode, um die Rolle der T-Zellen in einer Immunantwort zu beurteilen. Dieses Protokoll beschreibt die in Vitro Differenzierung von Knochenmark (BM) Stammväter Granulozyten Makrophagen Kolonie-stimulierende Faktor (GM-CSF) abgeleitet-Dendritische Zellen (DCs) zu erhalten. Das Protokoll beschreibt auch die Adoptiveltern Übertragung von Ovalbumin Peptid (OVAp)-GM-CSF-derived DCs und naive CD4-T-Zellen aus transgenen Mäusen OTII geladen, um die in-Vivo -Aktivierung, Verbreitung und Th1 Differenzierung zu analysieren die übertragenen CD4-T-Zellen. Dieses Protokoll umgeht die Begrenzung der rein in Vivo Methoden durch die Unfähigkeit, speziell zu manipulieren oder wählen die untersuchten Zellpopulation. Darüber hinaus ermöglicht dieses Protokolls Studien in einer in-Vivo -Umgebung, wodurch Veränderungen funktionale Faktoren, die in Vitro auftreten können sowie den Einfluss von Zelltypen und andere Faktoren, die nur in intakten Organe gefunden. Das Protokoll ist ein nützliches Werkzeug zur Erzeugung von Änderungen in DCs und T-Zellen, die adaptive Immunantworten, Bereitstellung von potenziell wichtige Ergebnisse zu verstehen, den Ursprung oder die Entwicklung von zahlreichen damit verbundenen Immunerkrankungen zu ändern.

Abbildung 1 zeigt die in diesem Protokoll beschriebenen Schritte. Abbildung 2 veranschaulicht die Isolation und die Kultur der BM Mauszellen. Die Zugabe von GM-CSF und LPS zu diesen Kulturen ermöglicht die in-vitro- Erzeugung und Reifung der DCs. Abbildung 3 veranschaulicht die Flow-zytometrie-Analyse der Differenzierung und Reifung der erhaltenen DCs. OVAp geladen GM-CSF BM abgeleitet DCS ...

Watch this Scientific Journal Video about An In Vivo Mouse Model to Measure NaÃ¯ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells at JoVE

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Hier präsentieren wir ein Protokoll zur in Vivo Bestimmung der naiven CD4-T Zellaktivierung (T Cell), Proliferation und Th1 Differenzierung induziert durch GM-CSF Knochenmark (BM)-abgeleitete Dendritische Zellen (DCs). Darüber hinaus beschreibt dieses Protokolls BM und T-Zellen isoliert, DC-Generation und DC und T-Zell-Adoptiv-Transfer.

Eine In Vivo Mausmodell Maßnahme naive CD4-T-Zell-Aktivierung, Proliferation und Th1 Differenzierung induziert durch Knochenmark-abgeleitete Dendritische Zellen

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

This method can help answer key questions in the immunology field about T-cell activation, proliferation, and Th1 differentiation, as well antigen presenting cell stimulation of adaptive immunity. The main advantage of this technique is that it allows the study of an in vivo environment while simultaneously allowing cell manipulation in vitro. Visual demonstration of this method is critical as the organ and bone isolation and adoptive transfer steps are difficult to master through text instructions alone.For bone marrow cell isolation, disinfect the hind limbs of an adult mouse with 70%ethanol and use scissors to make a lateral skin incision across the hind limbs. Insert scissors at the medial aspect of the legs, and cut the skin with the scissors until the beginning of the hind limbs. Then use forceps to separate the skin from the legs.Using scissors, separate the muscle from the femur and tibia, and use scissors to disarticulate the hip joint, breaking the femur head. Then separate the femure from the tibia without breaking the bone ends, and place the bones in a petri dish of complete medium on ice. When all of the bones have been harvested, carefully cut off the proximal and distal ends of each bone with a scalpel, and use a 1 millileter syringe equipped with a 25-guage needle to flush the bones repeatedly with a total volume of 10 millileters of warm complete RPMI medium.When all of the bones have been flushed, transfer the entire contents of the dish to a 50-millileter conical tube fitted with a 70-micrometer pore nylon web filter, and carefully dislodge any debris and cell conglomerates with gentle stirring and pipetting. Collect the bone marrow cells by centrifugation. Resuspend the pellet in one millileter of four-degree Celsius red blood cell lysis buffer with pipetting, and incubate the mix on ice for five minutes.At the end of the incubation, stop the lysis with 10 millileter of PVS, and resuspend the pellet in 25 millileters of complete medium for a second centrifugation. Then resuspend the cells in 5 millileters of fresh complete medium for counting, and centrifuge the cells again. For secondary lymphoid tissue cell isolation, collect the inguinal, axillary, brachial, cervical, and mesentaric lymph nodes, and spleen from OT-II transgenic mice, and transfer the secondary lymphoid tissues into a plastic petri dish containing 10 millileters of complete RPMI medium.When all of the tissues have been harvested, transfer the lymph nodes and spleen to a 70-micrometer cell strainer in a 50-millileter conical tube, and use a syringe plunger to homogenize the tissues. Flush the strainer with up to 15 millileters of medium, and spin down the cells by centrifugation. Then isolate the CD4-positive T cells by magnetic bead sorting, according to standard protocols.For in vivo activation of the naive OT-II CD4-positive T-cells, label the magnetic bead-isolated T-cells with vital cell tracer dye, according to standard protocols, and resuspend the labeled cells at a 10 to the sixth cells per 100 microliters of PBS concentration. Then adoptively transfer 100 microliters of cells per animal via the tail vein. Pre-warm the mice before the injection in order to ensure that the tail veins are enough dilated to allow delivery of entire cell volume.After 24 hours, deliver 10 to the fifth LPS-matured OVA peptide-loaded dendritic cells by subcutaneous injection into the foot pad of each T-cell injected animal. Two and five days after the adoptive challenge, harvest the poplitial lymph nodes from the recipient animals for tissue processing and T-cell isolation, as demonstrated. To analyze T-cell activation, stain day two-isolated cells with fluorescent antibodies against CD4, CD69, and CD25 to determine the percentage of CD64-positive, CD25-positive T-cells within the recipient CD4-positive population by flow cytometry.To assess T-cell proliferation, stain day five-isolated cells with the appropriate fluorescent antibodies against CD4, and analyze the decay of the vital cell tracer dye signal in the donor CD4-positive T-cell population by flow cytometry. To evaluate Th1 differentiation, plate four times 10 to the fifth day five-isolated T-cells per well in 200 microliters of complete medium, supplemented with ionomycin and PMA per well, in a 96-well plate. Then place the plate in a 37 degree Celsius cell culture incubator for six hours, adding Brefeldin A to each well to block cytokine secretion during the last four hours of the stimulation.At the end of the incubation, centrifuge the plate, and discard the supernatant by rapid inversion. Stain the cells with anti-CD4 antibody, followed by fixation in 100 microliters of 2%paraformaldehyde and 1%sucrose for 20 minutes at room temperature. Permeabilize the cells with 50 microliters of permeabilization buffer for 30 minutes at 4 degrees Celsius, followed by a centrifugation wash with 150 microliters of PBS per well.Next, stain for the intracellular cytokines of interest, according to standard protocols for 30 minutes at room temperature, followed by two centrifugation washes with 150 microliters of PBS supplemented with 1%saponin per well. Then, analyze the cells by flow cytometry. Flow cytometric analysis of GM-CSF bone marrow-derived dendritic cells after LPS stimulation, reveals an upregulation of maturation markers, such as MHCII, CD80, and CD86.Naive CD4 positive OT-II T-cells become activated after adoptive transfer to recipient animals by their upregulation of T-cell activation markers surface expression, and multiple rounds of cell division. Upon activation, the proliferating CD-positive OT-II T-cells demonstrate a Th1 phenotype, as demonstrated by their intracellular interferon gamma expression. Once mastered, this technique can be completed in eight hours if it's performed properly.While attempting this procedure, it's important to remember to prepare all the reagents and materials the day before the experiment. After its development, this technique paved the way for researchers in immunology to study dendritic cells and T-cells in mice. After watching this video, you should have a good understanding of how to isolate bone marrow from the femurs and tibia of mice, and how to transfer T-cells and dendritic cells to recipient animals.Don't forget that working with sharp instruments such needles, scalpels, or scissors, can be extremely hazardous, so precautions such as using the appropriate finger protection should always be taken while performing these procedures.

Research

JoVE Journal

Immunology and Infection

Retroviral Transduction of T-cell Receptors in Mouse T-cells

Retrovirale Transduktion von T-Zell-Rezeptoren in Maus-T-Zellen

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Forschung

Immunologie und Infektion

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Erzeugung und Kennzeichnung von Murine Knochenmark gewonnenen dendritischen Zellen mit Qdot Nanokristalle für Tracking-Studien

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Adenovirale Transduktion von Naive CD4 T-Zellen zu Treg Differenzierung Studieren

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

Isolierung und Differenzierung von Th17 Naive CD4-T-Lymphozyten

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Beurteilung der Innate Sensing von HIV-1-infizierten CD4<sup&gt; +</sup&gt; T-Zellen durch Plasmazytoide Dendritische Zellen unter Verwendung eines<em&gt; Ex-vivo-</em&gt; Co-Kultursystem.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

Isolierung und intravenöse Injektion von Maus-Knochenmark stammenden Monozyten

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Maus Naive CD4<sup&gt; +</sup&gt; T-Zell-Isolierung und<em&gt; In-vitro-</em&gt; Differenzierung in T-Zell-Populationen

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

T-Zellen erfassen Bakterien durch Transinfection von dendritischen Zellen

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Isolation und<em&gt; Ex Vivo</em&gt; Kultur des Vδ1<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt; γδ T-Zellen, eine extrathymic αβT-Zell-Vorläufer

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

In vitro Differenzierung von humanen CD4 + FOXP3 + Induced regulatorischer T - Zellen (iTregs) von Naïve CD4 + T - Zellen unter Verwendung eines TGF-β-enthaltende Protokoll