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

Ringraziamo il Dr. Simon Bartlett per l'editing di inglese. Questo studio è stato sostenuto da sovvenzioni da Instituto de Salud Carlos III (ISCIII) (PI14/00526; PI17/01395; CP11/00145; CPII16/00022), il programma di Miguel Servet e Fundación Ramón Areces; con il co-finanziamento del Fondo Europeo de Desarrollo Regional (FEDER). Il CNIC è supportato dal Ministero dell'economia, industria e competitività (MEIC) e la Fondazione Pro CNIC, è un Severo Ochoa Center of Excellence (SEV-2015-0505). RTF è fondata da Fundación Ramón Areces e CNIC, VZG da ISCIII, BHF di Instituto de Investigación Sanitaria Hospital 12 de Octubre (imas12) e JMG-G dal ISCIII Miguel Servet programma e imas12.

Procedure sperimentali sono state approvate dalla Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC) e la Comunidad Autónoma de Madrid conformemente agli orientamenti spagnoli ed europei. I topi sono stati allevati in condizioni di agente patogeno specifico libera (SPF) e sono stati eutanasizzati per inalazione di anidride carbonica (CO2).
1. isolamento delle cellule del midollo osseo del topo da tibie e femori
Nota: Il ceppo ...

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+envelope+lamin-A+couples+actin+dynamics+with+immunological+synapse+architecture+and+T+cell+activation.">Nuclear envelope lamin-A couples actin dynamics with immunological synapse architecture and T cell activation.</a> Science Signaling. 7 (322), ra37 (2014).</li><li>Rocha-Perugini, V., 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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Questo protocollo consente la caratterizzazione della capacità del DCs BM-derivato di modulare l'attivazione, proliferazione e differenziazione delle cellule T CD4 Naive. Inoltre, può anche essere utilizzato per valutare la suscettibilità delle cellule T CD4 + a modulazione da DCs BM-derivato. Con questo protocollo, le modifiche a questi eventi possono essere misurato in vivo.
Secondo l'ipotesi in esame, diverse combinazioni di cellule T e DC possono essere usati. Ad esempio, si po...

Le cellule T CD4 + e cellule (APCs) come DCs presentanti l'antigene sono necessari mediatori di immunità agli agenti patogeni microbici1,2. Negli organi linfoidi periferici, le cellule T CD4 sono attivate al momento della rilevazione di antigeni specifici presentati da APC3,4,5. Linfociti T CD4 attivati proliferano e si differenziano in cellule effettrici specifiche distinte del Th che sono necessari per lo sviluppo di una corretta risposta immunitaria adattativa6,7. Controllo di questi processi è fondamentale per la produzione di un'adeguata difesa adattiva che uccide l'agente patogeno senza produrre tessuto nocivo danni8. Cellule del Th sono definite secondo l'espressione o la produzione di molecole di superficie, fattori di trascrizione e le citochine effettrici e svolgono funzioni essenziali e precisione in risposta ad agenti patogeni1. Cellule del sottoinsieme delle cellule Th1 esprimono il fattore di trascrizione T-scommessa e la citochina interferone γ (IFNγ) e partecipano nella difesa ospite contro patogeni intracellulari1. Quantificazione dell'attivazione di cellule T CD4 Naive, proliferazione e differenziazione Th1 è un strumento utile per valutare il ruolo T cellule nella risposta immunitaria.
Questa protocollo consente in vivo analisi della capacità di in vitro -generato DCs BM-derivato di modulare l'attivazione, proliferazione e differenziazione Th1 delle cellule T CD4 Naive. Il protocollo serve anche a valutare la capacità delle cellule T CD4 Naive per essere attivato, indotti a proliferare e Th1 differenziato (Figura 1). Questo protocollo versatile elude l'incapacità di specificamente manipolare o selezionare la popolazione delle cellule studiate in puramente in vivo protocolli. Gli effetti di diverse molecole e trattamenti su controller di dominio possono essere studiati utilizzando BM da topi geneticamente modificati5 o trattamento o manipolare geneticamente isolata BM cellule9. Allo stesso modo, le risposte delle cellule T possono essere esplorate di ottenimento delle cellule di T per trasferimento adottivo da fonti diverse o dopo diverse manipolazioni3,8,10.
I principali vantaggi di questo protocollo sono duplici. L'attivazione delle cellule t, la proliferazione e la differenziazione Th1 sono analizzati con un approccio di citometria a flusso; e questo è combinato con studi in vivo , così evitare le alterazioni che possono verificarsi in vitro e compresi tipi cellulari e altri fattori solo trovati in organi intatti11.
L'uso di coloranti vitali è una tecnica ampiamente utilizzata per tenere traccia di proliferazione delle cellule, evitando l'uso di radioattività. La misurazione di proliferazione con questi reagenti si basa sulla diluizione della tintura dopo la divisione cellulare. Inoltre, questi coloranti possono essere rilevati a più lunghezze d'onda e facilmente sono analizzati tramite flusso cytometry in combinazione con più anticorpi fluorescenti o marcatori. Evidenziamo l'utilità del presente protocollo, mostrando come l'attivazione delle cellule T, la proliferazione e la differenziazione Th1 possono essere analizzati tramite flusso cytometry.

<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

Quantificazione dell'attivazione di cellule T CD4 Naive, la proliferazione e la differenziazione di T helper 1 (Th1) cellule è un modo utile per valutare il ruolo svolto dalle cellule di T in una risposta immunitaria. Questo protocollo descrive la differenziazione in vitro di progenitori del midollo osseo (BM) per ottenere granulocita macrofago distimolazione factor (GM-CSF) derivato-cellule dendritiche (DCs). Il protocollo descrive anche il trasferimento adottivo del peptide di ovoalbumina (OVAp)-caricato di GM-CSF-derivati DCs e cellule T CD4 Naive da topi transgenici Paolo al fine di analizzare l'attivazione in vivo , la proliferazione e la differenziazione Th1 della trasferite le cellule T CD4. Questo protocollo elude la limitazione di puramente in vivo metodi imposti dalla incapacità di specificamente manipolare o selezionare la popolazione delle cellule studiate. Inoltre, questo protocollo permette studi in un ambiente in vivo , evitando così alterazioni di fattori funzionali che possono verificarsi in vitro e compreso l'influenza di tipi cellulari e di altri fattori trovati solo negli organi intatti. Il protocollo è uno strumento utile per la generazione di cambiamenti nel sistema DCs e cellule di T che modificare le risposte immunitarie, potenzialmente fornire risultati importanti per capire l'origine o lo sviluppo di numerose patologie immuni.

La figura 1 illustra i passaggi descritti in questo protocollo. La figura 2 illustra l'isolamento e la coltura delle cellule BM del topo. L'aggiunta di GM-CSF e LPS a queste culture permette la generazione in vitro e maturazione della DCs. Figura 3 illustra l'analisi di citometria a flusso della differenziazione e maturazione della DCs. OVAp-caricato GM-CSF BM-derivato DCs ottenuti e isolato...

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

Qui, presentiamo un protocollo per la determinazione in vivo dell'ingenuo T CD4 delle cellule (cellule T) attivazione, proliferazione e differenziazione Th1, indotta da GM-CSF midollo osseo (BM)-derivato le cellule dendritiche (DCs). Inoltre, questo protocollo descrive isolamento BM e T-cell, generazione di DC e DC e T-cell trasferimento adottivo.

Un modello di topo In Vivo di attivazione delle cellule T Naïve CD4 di misura, la proliferazione e la differenziazione Th1 indotta dalle cellule dendritiche derivate da midollo osseo

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

Trasduzione retrovirale di recettori delle cellule T nel topo le cellule T

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 ...

Ricerca

Immunologia e infezioni

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

Generazione e l&#39;etichettatura di Murine osseo cellule dendritiche derivate dal midollo con nanocristalli Qdot per gli Studi inseguimento

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

Trasduzione Adenoviral di CD4 T naive Cells allo Studio Differenziazione Treg

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

Isolamento e differenziazione di Th17 Naïve linfociti T CD4

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.

Valutare l&#39;innato rilevamento dell&#39;infezione da HIV-1 CD4 infetti<sup&gt; +</sup&gt; Cellule T dalle cellule dendritiche plasmacitoidi Utilizzando un<em&gt; Ex vivo</em&gt; Sistema di co-coltura.

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

Isolamento e iniezione endovenosa di Murine derivate dal midollo osseo monociti

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

Mouse Naïve CD4<sup&gt; +</sup&gt; Isolamento T cellulare e<em&gt; In vitro</em&gt; Differenziazione in T sottopopolazioni cellulari

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

Cellule T Capture batteri da Transinfection da cellule dendritiche

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

Isolamento e<em&gt; Ex Vivo</em&gt; Cultura di Vδ1<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt; Celle γδ T, un Extrathymic αβT-cellule progenitrici

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

La differenziazione in vitro di cellule CD4 + umana FOXP3 + indotta da T regolatorie (iTregs) da cellule T CD4 Naïve utilizzando un protocollo TGF-β contenenti