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 NaÃƒÂ¯ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells at JoVE

Vi takker Dr. Simon Bartlett engelsk redigeringsspråk. Denne studien ble støttet av tilskudd fra Instituto de Salud Carlos III (ISCIII) (PI14/00526; PI17/01395; CP11/00145; CPII16/00022), Miguel Servet programmet og Fundación Ramón Areces; med co-finansiering fra Fondo Europeo de Desarrollo Regional (FEDER). CNIC er støttet av departementet for økonomi, industri og konkurranseevne (MEIC) og Pro CNIC stiftelsen og er en Severo Ochoa Center of Excellence (SEV-2015-0505). RTF er grunnlagt av Fundación Ramón Areces og CNIC, VZG av ISCIII, BHF ved Instituto de Investigación Sanitaria Hospital de 12 Octubre (imas12) og JMG-G ISCIII Miguel Servet programmet og imas12.

Eksperimentell prosedyrer ble godkjent av den Fundación Nacional Centro de Investigaciones Cardiovasculares Carlos III (CNIC) og Comunidad Autónoma de Madrid spanske og europeiske retningslinjer. Mus ble avlet i bestemte patogen gratis (SPF) forhold og ble euthanized av karbondioksid (CO2) innånding.
1. isolering av musen bein margtransplantasjon celler fra Tibias og Femurs
Merk: C57BL/6 congenic musen belastningen bærer differensial leukocytter markør...

<ol>
	<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. 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. M. <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+as+a+coordinator+of+T+cell+activation.">Nuclear envelope lamin-A as a coordinator of T cell activation.</a> Nucleus. 5 (5), 396-401 (2014).</li><li>Rocha-Perugini, V., 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=CD9+Regulates+Major+Histocompatibility+Complex+Class+II+Trafficking+in+Monocyte-Derived+Dendritic+Cells.">CD9 Regulates Major Histocompatibility Complex Class II Trafficking in Monocyte-Derived Dendritic Cells.</a> Molecular and Cellular Biology. 37 (15), (2017).</li><li>Kaech, S. M., Wherry, E. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Differentiation+of+Naive+CD4(+)+T+Cells:+A+Tool+for+Understanding+the+Development+of+Atherosclerosis.">In Vitro Differentiation of Naive CD4(+) T Cells: A Tool for Understanding the Development of Atherosclerosis.</a> Methods in Molecular Biology. 1339, 177-189 (2015).</li><li>Toribio-Fernandez, R., 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=Lamin+A/C+augments+Th1+differentiation+and+response+against+vaccinia+virus+and+Leishmania+major.">Lamin A/C augments Th1 differentiation and response against vaccinia virus and Leishmania major.</a> Cell Death &amp; Disease. 9 (1), 9 (2018).</li><li>Lee, T., Shevchenko, I., Sprouse, M. L., Bettini, M., Bettini, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+Transduction+of+Bone+Marrow+Progenitor+Cells+to+Generate+T-cell+Receptor+Retrogenic+Mice.">Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice.</a> Journal of Visualized Experiments. (113), (2016).</li><li>Cruz-Adalia, A., 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=Conventional+CD4(+)+T+cells+present+bacterial+antigens+to+induce+cytotoxic+and+memory+CD8(+)+T+cell+responses.">Conventional CD4(+) T cells present bacterial antigens to induce cytotoxic and memory CD8(+) T cell responses.</a> Nature Communications. 8 (1), 1591 (2017).</li><li>Quah, B. J., Wijesundara, D. K., Ranasinghe, C., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+fluorescent+target+arrays+for+assessment+of+T+cell+responses+in+vivo.">The use of fluorescent target arrays for assessment of T cell responses in vivo.</a> Journal of Visualized Experiments. (88), e51627 (2014).</li><li>Cruz-Adalia, A., Ramirez-Santiago, G., Torres-Torresano, M., Garcia-Ferreras, R., Veiga Chacon, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+Cells+Capture+Bacteria+by+Transinfection+from+Dendritic+Cells.">T Cells Capture Bacteria by Transinfection from Dendritic Cells.</a> Journal of Visualized Experiments. (107), e52976 (2016).</li><li>Cossarizza, A., 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=Guidelines+for+the+use+of+flow+cytometry+and+cell+sorting+in+immunological+studies.">Guidelines for the use of flow cytometry and cell sorting in immunological studies.</a> European Journal of Immunology. 47 (10), 1584-1797 (2017).</li><li>Quah, B. J., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+and+improved+methods+for+measuring+lymphocyte+proliferation+in+vitro+and+in+vivo+using+CFSE-like+fluorescent+dyes.">New and improved methods for measuring lymphocyte proliferation in vitro and in vivo using CFSE-like fluorescent dyes.</a> Journal of Immunological Methods. 379 (1-2), 1-14 (2012).</li><li>Yardeni, T., Eckhaus, M., Morris, H. D., Huizing, M., Hoogstraten-Miller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retro-orbital+injections+in+mice.">Retro-orbital injections in mice.</a> Lab Animals (NY). 40 (5), 155-160 (2011).</li><li>Warth, S. C., Heissmeyer, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral+transduction+of+naive+CD4+T+cells+to+study+Treg+differentiation.">Adenoviral transduction of naive CD4 T cells to study Treg differentiation.</a> Journal of Visualized Experiments. (78), (2013).</li><li>Liou, H. L., Myers, J. T., Barkauskas, D. S., Huang, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+the+mouse+popliteal+lymph+node.">Intravital imaging of the mouse popliteal lymph node.</a> Journal of Visualized Experiments. (60), (2012).</li></ol>

Denne protokollen gir karakterisering av kapasiteten til BM-avledet DCs å modulere aktivisering, spredning og differensiering av naiv CD4 T-celler. Videre kan det også brukes til å vurdere mottakelighet av CD4 T-celler for modulering av BM-avledet DCs. Med denne protokollen kan endringer i disse hendelsene målt i vivo.
Avhengig av hypotesen under etterforskning, kan flere kombinasjoner av T-celler og DCs brukes. For eksempel kan en analysere konsekvensene av banke i eller slå ut ...

CD4 T celler og antigen presentere celler (APCs) som DCs er nødvendig meglere av immunitet til mikrobiell patogener1,2. I perifere lymfoide organer, er CD4 T celler aktivert på anerkjennelse av bestemte antigener presentert av APCs3,4,5. Aktivert CD4 T celler spredning og differensiere i forskjellige bestemt effektor Th celler som er nødvendige for utviklingen av en riktig adaptive immunrespons6,7. Kontroll av disse prosessene er kritisk for å produsere en tilstrekkelig adaptive forsvar som dreper patogen uten å produsere skadelige vev skade8. Th celler defineres etter uttrykk eller produksjon av overflaten molekyler, transkripsjonsfaktorer og effektor cytokiner og utføre viktig og nøyaktige svar på patogener1. Celler Th1 celle delmengden express transkripsjon faktoren T-bet og cytokin interferon γ (IFNγ) og delta i vert forsvar mot intracellulær patogener1. Kvantifisering av naiv CD4 T celle aktivering og spredning Th1 differensiering er et nyttig vurdere T celler rollespill i en immunrespons.
Denne protokollen gir i vivo analyse av kapasiteten til in vitro -generert BM-avledet DCs å modulere aktivisering, spredning og Th1 differensiering av naiv CD4 T-celler. Protokollen fungerer også å vurdere kapasitet naiv CD4 T-celler aktivert, overtalt til å spre og Th1 differensiert (figur 1). Denne allsidige protokollen omgår kan spesielt manipulere eller velg studerte celle befolkningen i rent i vivo protokoller. Effekten av diverse molekyler og behandlinger på domenekontrollere kan studeres ved hjelp av BM genmodifiserte mus5 eller behandle eller genetisk manipulering isolert BM celler9. Tilsvarende kan T celle svar utforskes ved å skaffe T celler for adopsjon overføring fra ulike kilder eller etter flere manipulasjoner3,8,10.
De viktigste fordelene med denne protokollen er todelt. T celle aktivering og spredning Th1 differensiering analyseres med en flyt cytometri tilnærming; og dette er kombinert med i vivo studier, dermed avverge endringer som kan oppstå i vitro og inkludert celletyper og andre faktorer som bare finnes i intakt organer11.
Bruk av vitale fargestoffer er en mye brukt teknikk å spore celle spredning og unngå bruk av radioaktivitet. Måling av spredning med disse reagensene er basert på fargestoff fortynning etter celledeling. Videre disse fargestoffer kan oppdages ved flere bølgelengder og analyseres lett av flowcytometri i kombinasjon med flere fluorescerende antistoffer eller markører. Vi markere nytten av denne protokollen ved å vise hvordan T celle aktivering og spredning Th1 differensiering kan analyseres av flowcytometri.

<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

Kvantifisering av naiv CD4 T celle aktivering, spredning og differensiering å T helper 1 (Th1) celler er en nyttig måte å vurdere rollen spilt av T-celler i en immunrespons. Denne protokollen beskriver i vitro differensiering av benmarg (BM) progenitors hente granulocytt-macrophage koloni-stimulerende faktor (GM-CSF) avledet-dendrittiske celler (DCs). Protokollen beskriver også adoptivforeldre overføring av ovalbumin peptid (OVAp)-lastet GM-CSF-avledet DCs og naiv CD4 T celler fra OTII transgene mus for å analysere den i vivo aktivering, spredning og Th1 differensiering av den overførte CD4 T-celler. Denne protokollen omgår begrensning av rent i vivo metoder pålagt av kan spesielt manipulere eller velg studerte celle befolkningen. Denne protokollen gir videre studier i i vivo miljø, dermed unngår endringer til funksjonelle faktorer som kan oppstå i vitro og med påvirkning av celletyper og andre faktorer som bare finnes i intakt organer. Protokollen er et nyttig verktøy for å generere endringer i DCs og T celler som endre adaptive immunreaksjoner, å gi viktige resultater for å forstå opprinnelsen og utviklingen av mange immun tilknyttede sykdommer.

Figur 1 illustrerer fremgangsmåten beskrevet i denne protokollen. Figur 2 viser den isolasjon og kultur av musen BM celler. Tillegg av GM-CSF og LP-plater til disse kulturene lar den i vitro generasjonen og modning av DCs. Figur 3 illustrerer flyten cytometri analyse av differensieringen og modningen av de fått DCs. OVAp lastet GM-CSF BM-avledet DCs og isolert viktig celle spor fargestoff-...

Watch this Scientific Journal Video about An In Vivo Mouse Model to Measure NaÃƒÂ¯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 NaÃƒÆ’Ã†'Ãƒâ€šÃ‚Â¯ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Her presenterer vi i vivo fastsettelse av naiv CD4 T celle (T celle) aktivering, spredning og Th1 differensiering av GM-CSF benmargen (BM) er en protokoll-avledet dendrittiske celler (DCs). I tillegg beskriver denne protokollen BM og T-celle isolasjon, DC generasjon og DC og T-celle adoptivforeldre overføring.

En i Vivo musemodell mål naiv CD4 T celle aktivering, spredning og Th1 differensiering av Ben margtransplantasjon-avledet dendrittiske celler

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 ...

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JoVE Journal

Immunology and Infection

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

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

Vurdere Medfødt Sensing av HIV-1 infiserte CD4<sup&gt; +</sup&gt; T-celler ved Plasmacytoid dendrittiske celler ved hjelp av en<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 ...

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