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

Wij danken Dr. Simon Bartlett voor Engelse bewerken. Deze studie werd ondersteund door subsidies van het Instituto de Salud Carlos III (ISCIII) (PI14/00526; PI17/01395; CP11/00145; CPII16/00022), de Miguel Servet programma- en Fundación Ramón Vicente Areces; met co-financiering van de Fondo Europeo de Desarrollo Regional (EFRO). De CNIC wordt ondersteund door het ministerie van economische zaken, industrie en concurrentievermogen (MEIC) en de Pro CNIC Foundation en kan een Severo Ochoa Center of Excellence (SEV-2015-0505). RTF is opgericht door Fundación Ramón Vicente Areces en CNIC, VZG door ISCIII, BHF door Instituto de Investigación Sanitaria ziekenhuis 12 de Octubre (imas12) en JMG-G door de ISCIII Miguel Servet programma en imas12.

Experimentele procedures werden goedgekeurd door de Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC) en de Comunidad Autónoma de Madrid overeenkomstig de Spaanse en Europese richtsnoeren. Muizen werden gefokt in specifieke pathogene vrije (SPF) voorwaarden en werden euthanized kooldioxide (CO2) inademing.
1. isolatie van muis beenmergcellen van zijn verbeend en dijbeen
Opmerking: De C57BL/6 congenisch muis stam draagt de dif...

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	<li>Zhu, J., Yamane, H., Paul, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+effector+CD4+T+cell+populations+(*).">Differentiation of effector CD4 T cell populations (*).</a> Annual Review of Immunology. 28, 445-489 (2010).</li><li>Bustos-Moran, E., Blas-Rus, N., Martin-Cofreces, N. B., Sanchez-Madrid, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orchestrating+Lymphocyte+Polarity+in+Cognate+Immune+Cell-Cell+Interactions.">Orchestrating Lymphocyte Polarity in Cognate Immune Cell-Cell Interactions.</a> International Review of Cell and Molecular Biology. 327, 195-261 (2016).</li><li>Gonzalez-Granado, J. 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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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Dit protocol zorgt voor de karakterisering van de capaciteit van BM afkomstige DCs aan het moduleren van de activering, proliferatie en differentiatie van naïeve CD4 T-cellen. Bovendien, het kan ook worden gebruikt om te beoordelen van de gevoeligheid van CD4 T-cellen voor modulatie door BM afkomstige DCs. Met dit protocol, kunnen veranderingen in deze gebeurtenissen worden gemeten in vivo.
Afhankelijk van de hypothese onderzochte, kunnen verschillende combinaties van T-cellen en DCs...

CD4 T-cellen en antigeen presentatie van cellen (APCs) zoals DCs zijn vereist bemiddelaars van immuniteit aan microbiële ziekteverwekkers1,2. In perifere lymfoïde organen, worden CD4 T-cellen geactiveerd bij opname van specifieke antigenen gepresenteerd door APCs3,4,5. Geactiveerde CD4 T-cellen vermenigvuldigen en onderscheid maken in verschillende specifieke effector Th-cellen die nodig voor de ontwikkeling van een juiste adaptieve immuunrespons6,7 zijn. Controle van deze processen is cruciaal voor het produceren van een adequate adaptieve verdediging die het pathogene agens oplevert doodt zonder schadelijke weefsel schade8produceren. Th-cellen zijn gedefinieerd volgens de uitdrukking of de productie van oppervlakte moleculen, transcriptiefactoren en effector cytokines en essentieel en precieze functies vervullen in antwoord op ziekteverwekkers1. Cellen van de Th1 cel subset express de transcriptiefactor T-inzet en de cytokine interferon-γ (IFNγ) en host verdediging tegen intracellulaire pathogenen1deelnemen. Kwantificering van naïeve CD4 T-cel activatie, proliferatie en differentiatie van de Th1 is een nuttig instrument voor de beoordeling van de rol van T cellen spelen in een immuunrespons.
Dit protocol maakt het mogelijk in vivo analyse van de capaciteit van in vitro -gegenereerd BM afkomstige DCs aan het moduleren van de activering, proliferatie en differentiatie van de Th1 van naïeve CD4 T-cellen. Het protocol dient ook te beoordelen van de capaciteit van naïeve CD4 T-cellen worden geactiveerd, geïnduceerde te verspreiden, en Th1 gedifferentieerde (Figuur 1). Dit veelzijdige protocol omzeilt het onvermogen om te specifiek bewerken of selecteer de bestudeerde celpopulatie in zuiver in vivo protocollen. De effecten van verschillende moleculen en behandelingen op DCs kunnen bestudeerd worden met behulp van BM van genetisch gemodificeerde muizen5 of behandelen of geïsoleerde BM cellen9genetisch te manipuleren. T cel reacties kunnen ook worden onderzocht door het verkrijgen van T-cellen voor de overdracht van het adoptie van verschillende bronnen of na verschillende manipulaties3,-8,10.
De belangrijkste voordelen van dit protocol zijn tweeledig. T-cel activatie, proliferatie en differentiatie van de Th1 worden geanalyseerd met een stroom cytometry benadering; en dit wordt gecombineerd met in vivo studies, dus het afwenden van wijzigingen die in vitro kunnen optreden en met inbegrip van celtypes en andere factoren die alleen gevonden in intact organen11.
Het gebruik van vitale kleurstoffen is een veel gebruikte techniek voor het bijhouden van celproliferatie terwijl het vermijden van het gebruik van radioactiviteit. De meting van de proliferatie met deze reagentia is gebaseerd op de kleurstof verdunning na de celdeling. Bovendien, deze kleurstoffen op meerdere golflengten gedetecteerd kunnen worden en gemakkelijk worden geanalyseerd door stroom cytometry in combinatie met meerdere fluorescerende antilichamen of markeringen. Wij wijzen op het nut van dit protocol door aan te tonen hoe T-cel activatie, proliferatie en differentiatie van de Th1 door stroom cytometry kunnen worden geanalyseerd.

<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

Kwantificering van naïeve CD4 T-cel activatie, proliferatie en differentiatie naar T-helper 1 (Th1) cellen is een handige manier om te beoordelen van de rol van T-cellen in een immuunrespons. Dit protocol beschrijft de in vitro differentiatie van het beenmerg (BM) progenitoren te verkrijgen granulocyt macrofaag kolonie-stimulerende factor (GM-CSF) afgeleid-dendritische cellen (DC's). Het protocol beschrijft ook de adoptief overdracht van ovoalbumine peptide (OVAp)-geladen GM-CSF-afgeleide DCs en naïef CD4 T-cellen van th transgene muizen om te analyseren de in vivo activering, proliferatie en Th1 differentiatie van de overgedragen CD4 T-cellen. Dit protocol omzeilt de beperking van zuiver in vivo methoden opgelegd door het onvermogen om specifiek manipuleren of selecteer de bestudeerde celpopulatie. Bovendien kan dit protocol studies in een in vivo omgeving, dus het vermijden van veranderingen aan functionele factoren die zich in vitro voordoen kunnen en met inbegrip van de invloed van celtypes en andere factoren die alleen gevonden in intact organen. Het protocol is een nuttig instrument voor het genereren van wijzigingen in DCs en T cellen die wijzigen van adaptieve immuunresponsen, mogelijk het verstrekken van belangrijke resultaten om te begrijpen van de oorsprong of de ontwikkeling van talloze immuun geassocieerde ziekten.

Figuur 1 illustreert de stappen die worden beschreven in dit protocol. Figuur 2 illustreert de isolatie en de cultuur van muis BM cellen. De toevoeging van GM-CSF en LP's aan deze culturen maakt de in vitro generatie en rijping van DCs. Figuur 3 illustreert de stroom cytometry analyse van de differentiatie en rijping van de verkregen DCs. OVAp-geladen GM-CSF BM afkomstige DCs en geïsoleerde...

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 presenteren we een protocol voor de in vivo bepaling van naïeve CD4 T cel (T-cel) activering, proliferatie en differentiatie van de Th1 geïnduceerd door GM-CSF beenmerg (BM)-afgeleide dendritische cellen (DC's). Dit protocol beschrijft Daarnaast BM en T-cel isolatie, DC generatie en DC en T-cel adoptief overdracht.

Een muismodel In Vivo maatregel naïef CD4 T-cel activatie, proliferatie en differentiatie van de Th1 geïnduceerd door het beenmerg-afgeleide dendritische cellen

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 Transductie van T-cel-receptoren in Mouse T-cellen

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 ...

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

Generatie en etikettering van Muriene Bone Marrow-afgeleide dendritische cellen met Qdot Nanokristallen voor Tracking Studies

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 transductie van Naïeve CD4 T-cellen aan Treg Differentiatie Studie

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

Isolatie en Th17 Differentiatie van naïeve CD4 T-lymfocyten

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.

Het beoordelen van de Aangeboren Sensing van HIV-1 geïnfecteerde CD4<sup&gt; +</sup&gt; T cellen door plasmacytoïde dendritische cellen gebruiken een<em&gt; Ex vivo</em&gt; Co-cultuur systeem.

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

Isolatie en intraveneuze injectie van muizen beenmerg afgeleide Monocyten

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

Muis Naïve CD4<sup&gt; +</sup&gt; T Cell Isolatie en<em&gt; In vitro</em&gt; Differentiatie in T cel subsets

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-cellen Capture Bacteriën door Transinfection van dendritische cellen

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

Isolatie en<em&gt; Ex Vivo</em&gt; Cultuur van Vδ1<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt; γδ T-cellen, een Extrathymic αβT-cel Progenitor

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 differentiatie van humane CD4 + FOXP3 + geïnduceerde regulatoire T-cellen (iTregs) van naïeve CD4 + T cellen met behulp van een TGF-β-bevattende Protocol