Name: in vitro differentiation of mouse granulocyte-macrophage-colony-stimulating factor (gm-csf)-producing t helper (thgm) cells
Uploaded: 2018-09-10T14:00:00
Description: Watch this Scientific Journal Video about In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells at JoVE.com

Denne undersøgelse blev støttet af tilskud fra National University Health System of Singapore (T1-2014 okt-12 og T1-2015 Sep-10).

Alle mus bruges i denne protokol var på C57BL/6 genetiske baggrund og har til huse under specifikt patogenfrie betingelser på National University of Singapore. Alle eksperimenter blev udført ved hjælp af protokollerne er godkendt af institutionelle Animal Care og brug Udvalget af National University of Singapore.
1. reagens og materielle forberedelse
<ol><li>Forberede 500 mL af fosfatbufferet saltopløsning (PBS) med 2% føtal bovint serum (FBS) og 1 mM ethylendiamintetra syre (EDTA).<br...

<ol>
	<li>Zhu, J., 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=CD4+T+cells:+fates,+functions,+and+faults.">CD4 T cells: fates, functions, and faults.</a> Blood. 112, 1557-1569 (2008).</li><li>Kara, E. E., 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=Tailored+immune+responses:+novel+effector+helper+T+cell+subsets+in+protective+immunity.">Tailored immune responses: novel effector helper T cell subsets in protective immunity.</a> PLoS Pathogens. 10, e1003905 (2014).</li><li>Bou Nasser Eddine, F., Ramia, E., Tosi, G., Forlani, G., Accolla, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+Immunology+meets...Immunology:+Modified+cancer+cells+as+professional+APC+for+priming+naive+tumor-specific+CD4++T+cells.">Tumor Immunology meets...Immunology: Modified cancer cells as professional APC for priming naive tumor-specific CD4+ T cells.</a> Oncoimmunology. 6, e1356149 (2017).</li><li>Dong, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TH17+cells+in+development:+an+updated+view+of+their+molecular+identity+and+genetic+programming.">TH17 cells in development: an updated view of their molecular identity and genetic programming.</a> Nature Reviews. Immunology. 8, 337-348 (2008).</li><li>Korn, T., Bettelli, E., Oukka, M., Kuchroo, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-17+and+Th17+Cells.">IL-17 and Th17 Cells.</a> Annual Review of Immunology. 27, 485-517 (2009).</li><li>Sheng, W., 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=STAT5+programs+a+distinct+subset+of+GM-CSF-producing+T+helper+cells+that+is+essential+for+autoimmune+neuroinflammation.">STAT5 programs a distinct subset of GM-CSF-producing T helper cells that is essential for autoimmune neuroinflammation.</a> Cell Research. 24, 1387-1402 (2014).</li><li>Codarri, L., 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=RORgammat+drives+production+of+the+cytokine+GM-CSF+in+helper+T+cells,+which+is+essential+for+the+effector+phase+of+autoimmune+neuroinflammation.">RORgammat drives production of the cytokine GM-CSF in helper T cells, which is essential for the effector phase of autoimmune neuroinflammation.</a> Nature Immunology. 12, 560-567 (2011).</li><li>El-Behi, 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=The+encephalitogenicity+of+T(H)17+cells+is+dependent+on+IL-1-+and+IL-23-induced+production+of+the+cytokine+GM-CSF.">The encephalitogenicity of T(H)17 cells is dependent on IL-1- and IL-23-induced production of the cytokine GM-CSF.</a> Nature Immunology. 12, 568-575 (2011).</li><li>Ivanov, I. I., 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=The+orphan+nuclear+receptor+RORgammat+directs+the+differentiation+program+of+proinflammatory+IL-17++T+helper+cells.">The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells.</a> Cell. 126, 1121-1133 (2006).</li><li>Zhang, J., 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=A+novel+subset+of+helper+T+cells+promotes+immune+responses+by+secreting+GM-CSF.">A novel subset of helper T cells promotes immune responses by secreting GM-CSF.</a> Cell Death and Differentiation. 20, 1731-1741 (2013).</li><li>Croxford, A. L., Spath, S., Becher, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GM-CSF+in+Neuroinflammation:+Licensing+Myeloid+Cells+for+Tissue+Damage.">GM-CSF in Neuroinflammation: Licensing Myeloid Cells for Tissue Damage.</a> Trends in Immunology. 36, 651-662 (2015).</li></ol>

Her beskrev vi en protokol for en in vitro- THGM differentiering fra mus naive CD4 + celler, efterfulgt af en analyse af de differentierede celler til at validere metoden. Af note, kan både milt og lymfeknuder bruges til naive CD4 + T-celle rensning og THGM differentiering. Cytokin udtryk bestemmes af intracellulære cytokin farvning kombineret med flowcytometri viste, at omkring 55% af celler blev tilskyndet til at blive GM-CSF-udtrykker celler under THGM tilstand (<strong clas...

CD4 + T hjælper (TH) celler er væsentlige elementer af immunforsvaret, at have afgørende roller i vært forsvaret mod mikrobielle patogener, kræft overvågning og autoimmunitet1,2,3. Ved T-celle receptoren (TCR) aktivering, naive CD4 + T celler kan opdeles i TH1, TH2, TH 17, eller regulerende T (langsomme) celler under indflydelse af forskellige cytokin miljøer2,4, 5. for nylig en ny delmængde af TH celler, som hovedsageligt producerer GM-CSF, blev identificeret og navngivne THGM6. Differentiering af THGM celler er drevet af IL-7 gennem aktivering af en signal transducer og en aktivator af transskription 5 (STAT5). Disse celler udtrykker en stor mængde af GM-CSF og samtidig have et lavt udtryk for andre TH-celle signatur cytokiner såsom IFNγ og IL-176. GM-CSF blev fundet til at spille en afgørende rolle i udviklingen af CD4 + T-celle-medieret neuroinflammation7,8. I forhold til IFNγ - eller IL-17-udtrykker autoreactive T celler, celler GM-CSF-udtrykker T er overført til wild-type (WT) mus forårsaget en tidligere sygdom udbrud og højere sygdommens sværhedsgrad. Derudover mislykkedes Csf2- / - T celler at fremkalde eksperimentelle autoimmune encephalomyelitis (EAE) efter adoptively overført til WT modtagere, mens T celler mangler IFNγ eller IL-17A bevaret evnen til at mægle EAE7. Desuden forbedret en blokade af GM-CSF ved hjælp af neutraliserende antistoffer EAE sygdom sværhedsgraden8. Desuden, mangel af STAT5 i T-celler i mus resulterede i en formindsket THGM generation og dermed i en modstand af mus til EAE udvikling6. Disse resultater understreger betydningen af GM-CSF-udtrykker TH celler i autoimmune neuroinflammatory sygdom. Således ville indføres en metode til at adskille GM-CSF-udtrykker TH celler fra naiv CD4 + T celler være vigtigt i studiet af patogenesen af autoimmune neuroinflammation og T-celle-medieret immunrespons. Dog en protokol der effektivt genererer THGM celler fra murine naive CD4 + ikke er blevet etableret.
Her præsenterer vi en metode, der adskiller murine THGM celler fra naiv CD4 + T-celler. Denne protokol beskriver hele proceduren, herunder udvinding af milten fra musen, udarbejdelse af en enkelt celle suspension, CD4 positive udvælgelse, fluorescens-aktiveret celle sortering (FACS) og cellen TH differentiering og analyse. De differentierede T helper celler er analyseret af intracellulære cytokin farvning kombineret med flowcytometri at bestemme cytokin udtryk på én celle-plan af en kvantitativ real-time PCR til at bestemme cytokin udtryk på mRNA niveau, og ved ELISA at vurdere cytokin udtryk på protein niveau. Denne metode kan anvendes til undersøgelser af THGM cellebiologi under forskellige forhold, såsom EAE, hvor GM-CSF spiller en vigtig rolle i patogenesen.

<table>
	<tbody>
		<tr>
			<td>RPMI 1640</td>
			<td>Biowest</td>
			<td>L0500-500</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS Heat Inactivated</td>
			<td>Capricorn</td>
			<td>FBS-HI-12A</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>1st Base</td>
			<td>BUF-2040-10</td>
			<td>Diluted to 1x PBS in sterile water</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>1st Base</td>
			<td>BUF-1053-100ml-pH8.0</td>
			<td></td>
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		<tr>
			<td>10X ACK buffer</td>
			<td>Biolegend</td>
			<td>420301</td>
			<td>diluted in sterile water</td>
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		<tr>
			<td>cell strainer</td>
			<td>SPL Life Sciences</td>
			<td>93070</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4 microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-049-201</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M7522</td>
			<td></td>
		</tr>
		<tr>
			<td>LS column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>magnetic stand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>1ml syringe</td>
			<td>Terumo</td>
			<td>SS+01T</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>Eppendorf 5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>Sony SY3200 cell sorter</td>
			<td>Sony</td>
			<td>Sony SY3200 cell sorter</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml conical centrifuge tube</td>
			<td>Greiner Bio-One</td>
			<td>210261</td>
			<td></td>
		</tr>
		<tr>
			<td>15m conical centrifuge tube</td>
			<td>Greiner Bio-One</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tube</td>
			<td>Corning</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well cell culture plate</td>
			<td>Greiner Bio-One</td>
			<td>662160</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD4 PerCP-eFluor 710</td>
			<td>eBioscience</td>
			<td>46-0041-82</td>
			<td></td>
		</tr>
		<tr>
			<td>PE conjugated anti-mouse CD25 (IL-2Ra, p55)</td>
			<td>eBioscience</td>
			<td>12-0251-82</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human/mouse CD44 APC</td>
			<td>eBioscience</td>
			<td>17-0441-82</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD62L FITC</td>
			<td>eBioscience</td>
			<td>11-0621-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer-improved counting chamber</td>
			<td>Marienfeld</td>
			<td>640010</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyclone Trypan Blue Solution</td>
			<td>GE healthcare Life Sciences</td>
			<td>SV30084.01</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope</td>
			<td>Nikon</td>
			<td>Nikon Elipse TS100</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified&#160;anti-mouse CD3e&#160;antibody</td>
			<td>Biolegend</td>
			<td>100314</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified hamster anti-mouse CD28</td>
			<td>BD Biosciences</td>
			<td>553295</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified Rat&#160;anti-mouse IFN&#947; &#160;</td>
			<td>eBioscience</td>
			<td>16-7312-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified anti-mouse IL-4 Antibody</td>
			<td>Biolegend</td>
			<td>504102</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse IL-7 Protein</td>
			<td>R&#38;D system</td>
			<td>407-ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse IL-6</td>
			<td>R&#38;D system</td>
			<td>406&#8209;ML</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant human TGF-beta</td>
			<td>R&#38;D system</td>
			<td>240-B-010</td>
			<td></td>
		</tr>
		<tr>
			<td>PMA</td>
			<td>Merck Millipore</td>
			<td>19-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>Sigma</td>
			<td>I0634</td>
			<td></td>
		</tr>
		<tr>
			<td>GolgiPlug protein transport inhibitor</td>
			<td>BD Biosciences</td>
			<td>51-2301KZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Intracellular Fixation&#38;Permeabilization buffer set</td>
			<td>eBioscience</td>
			<td>88-8824-00</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse GM-CSF PE</td>
			<td>eBioscience</td>
			<td>12-7331-82</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse IL-17A</td>
			<td>Biolegend</td>
			<td>506908</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse IFN-gamma</td>
			<td>Biolegend</td>
			<td>505810</td>
			<td></td>
		</tr>
		<tr>
			<td>GO Taq qPCR master mix</td>
			<td>Promega</td>
			<td>A6002</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse GM-CSF ELISA Ready-SET-Go!</td>
			<td>invitrogen</td>
			<td>88-7334-88</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IL-17A Uncouted ELISA</td>
			<td>invitrogen</td>
			<td>88-7371-22</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro differentiation of mouse granulocyte-macrophage-colony-stimulating factor (gm-csf)-producing t helper (thgm) cells

Granulocyt-makrofag-koloni-stimulerende faktor (GM-CSF)-producerer T helper (THGM) celle er en nyligt identificerede T hjælper celle delmængde, som overvejende udskiller GM-CSF uden produktion af interferon (IFN) γ eller interleukin (IL) -17 og er fundet spil en væsentlig rolle i den autoimmune neuroinflammation. En metode til dyrkning af naive CD4 + T celler fra en enkelt celle suspension af splenocytes og THGM celle generation fra naiv CD4 + T celler ville være en nyttig teknik i studiet af T celle-medieret immunitet og autoimmune sygdomme. Her beskriver vi en metode, der adskiller mus naive CD4 + T-celler i THGM celler fremmes af IL-7. Resultatet af differentieringen blev vurderet ved analyse af cytokiner udtryk ved hjælp af forskellige teknikker, herunder intracellulære cytokin farvning kombineret med flowcytometri, en kvantitativ real-time polymerase kædereaktion (PCR), og enzym-forbundet immunosorbent assays (ELISA). Bruger THGM differentiering protokol som beskrevet her, udtrykt omkring 55% af cellerne GM-CSF med en minimal udtryk for IFNα eller IL-17. Den fremherskende udtryk af GM-CSF ved THGM celler blev yderligere bekræftet af analysen af udtryk af GM-CSF, IFNα og IL-17 på både mRNA og protein niveauer. Således, denne metode kan bruges til at differentiere naive CD4 + T-celler til THGM celler in vitro-, som vil være nyttige i studiet af THGM cellebiologi.

Naive CD4 + T celler isoleret fra to 8-uge-forhenværende mandlige C57BL/6 mus var opdelt i tre dele. En del af cellerne var opdeles i THGM celler efter protokollen beskrevet. En anden del blev kulturperler under en THGM betingelse i nærværelse af anti-IL-4 antistof (10 µg/mL) for at teste indflydelse af en IL-4 blokade i differentieringen af THGM. Den sidste del var kulturperler under TH17 differentiering betingelse (3 µg/mL anti-CD3e, 1 µ...

Watch this Scientific Journal Video about In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells at JoVE.com

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

In Vitro Differentiering af mus granulocyt-makrofag-koloni-stimulerende faktor (GM-CSF)-producerer T hjælper (THGM) celler

Vi præsenterer her, en protokol for at differentiere murine granulocyte-macrophage-colony-stimulating-factor-producing T hjælper (THGM) celler fra naiv CD4 + T celler, herunder isolering af naive CD4 + T celler, differentiering af THGM, og analyse af differentierede THGM celler. Denne metode kan anvendes til undersøgelser af forordningen og funktion af THGM celler.

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly ...

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In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

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