Name: isolating lymphocytes from the mouse small intestinal immune system
Uploaded: 2018-02-28T15:00:00
Description: Watch this Scientific Journal Video about Isolating Lymphocytes from the Mouse Small Intestinal Immune System at JoVE.com

B.S.S. understøttes af NIH grant (R01 AI076457) og midler fra Stony Brook University. Z.Q. understøttes af NIH yde (K12 GM102778).

Alle dyreforsøg blev gennemført i overensstemmelse med National Institute of Health retningslinjer og godkendt af Stony Brook University institutionelle Animal Care og brug udvalget.
Bemærk: Sikre, at alle godkendelser er tildelt før den udfører procedurer.
1. løsning forberedelse
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HGPG (HEPES, L-glutamin, penicillin/streptomycin og phosphatbufferet), 100 ×
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<li>Bland 59.6 g HEPES (500 mM endelige), 14,6 g L-glutamin (200 mM en...

<ol>
	<li>Hooper, L. V., Macpherson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+adaptations+that+maintain+homeostasis+with+the+intestinal+microbiota.">Immune adaptations that maintain homeostasis with the intestinal microbiota.</a> Nat Rev Immunol. 10 (3), 159-169 (2010).</li><li>Brandtzaeg, P., Kiyono, H., Pabst, R., Russell, M. 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(111), (2016).</li><li>Koscso, B., Bogunovic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+and+Purification+of+Mouse+Intestinal+Dendritic+Cell+and+Macrophage+Subsets+by+Flow+Cytometry.">Analysis and Purification of Mouse Intestinal Dendritic Cell and Macrophage Subsets by Flow Cytometry.</a> Curr Protoc Immunol. 114, 11-14 (2016).</li><li>Goodman, T., Lefrancois, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+gamma-delta+T-cell+receptor+on+intestinal+CD8++intraepithelial+lymphocytes.">Expression of the gamma-delta T-cell receptor on intestinal CD8+ intraepithelial lymphocytes.</a> Nature. 333 (6176), 855-858 (1988).</li><li>Huleatt, J. W., Lefrancois, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta2+integrins+and+ICAM-1+are+involved+in+establishment+of+the+intestinal+mucosal+T+cell+compartment.">Beta2 integrins and ICAM-1 are involved in establishment of the intestinal mucosal T cell compartment.</a> Immunity. 5 (3), 263-273 (1996).</li><li>Sheridan, B. S., 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=gammadelta+T+cells+exhibit+multifunctional+and+protective+memory+in+intestinal+tissues.">gammadelta T cells exhibit multifunctional and protective memory in intestinal tissues.</a> Immunity. 39 (1), 184-195 (2013).</li><li>Sheridan, B. 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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=The+lymph+nodes+draining+the+small+intestine+and+colon+are+anatomically+separate+and+immunologically+distinct.">The lymph nodes draining the small intestine and colon are anatomically separate and immunologically distinct.</a> Mucosal Immunol. 9 (2), 468-478 (2016).</li><li>Lorenz, R. G., Newberry, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+lymphoid+follicles+can+function+as+sites+for+induction+of+mucosal+immune+responses.">Isolated lymphoid follicles can function as sites for induction of mucosal immune responses.</a> Ann N Y Acad Sci. 1029, 44-57 (2004).</li><li>Kim, S. K., Reed, D. S., Heath, W. R., Carbone, F., Lefrancois, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+and+migration+of+CD8+T+cells+in+the+intestinal+mucosa.">Activation and migration of CD8 T cells in the intestinal mucosa.</a> J Immunol. 159 (9), 4295-4306 (1997).</li><li>Huleatt, J. 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En detaljeret protokol præsenteres til isolering af lymfocytter fra tarmens slimhinde induktiv (MLN og PP) og effektor (LP og IEL rum) websteder. Protokollen er udviklet til at skabe balance mellem input (tid) og output (rentabilitet og udbytte) til at maksimere produktiviteten og resultater. Protokollen også sikrer minimal afdelingsmæssig krydskontaminering mellem LP og IEL rum.
Flere protokoller til isolering af immunceller fra musen tyndtarmen har været udgivet3<...

Den gastrointestinale (GI) tarmkanalen har mange folder og fremspring, der repræsenterer den største grænseflade adskille den indre krop og det ydre miljø. Tarm immunforsvaret spiller en væsentlig rolle i at opretholde den barriere funktion af mave-tarmkanalen. Det er konstant udsat for kosten antigener, commensal bakterier og patogene mikrober. Som sådan må det forblive tolerant over for fødevarer antigener og commensal bakterier samtidig bevare evnen til at generere hurtigt en effektiv immunrespons på enteropathogenic mikrober1. Tarm immunsystemet kan anatomisk opdeles i induktive sites, hvor naiv lymfocytter aktiveres af antigen præsentere celler transporterer antigener fra tarmslimhinden, og effektor websteder, hvor aktiverede lymfocytter udøve bestemte effektor funktion2. De induktive sites omfatter organiseret lymfoide strukturen af Peyerske plaques (PP), undersøgelser af intestinal lumen direkte gennem handling af specialiserede M celler og regionale afdrypning mesenteriallymfeknuderne lymfeknuder (mio.). Effektor websteder består af lamina propria (LP), som er bindevæv under basalmembranen og den intestinale epitel, et enkelt cellelag placeret over basalmembran, der indeholder intraepithelial lymfocytter (IEL). Lymfocytter er store spillere af adaptive immunitet, der mægle beskyttelse mod infektioner og kræftformer og kan også bidrage til immunopathology i inflammatoriske sygdomme. Det er vigtigt og yderst relevant at studere lymfocytter i disse forskellige anatomiske slimhinde rum for bedre at kunne forstå deres induktion og effektor funktion.
En relativt enkel og samlet protokol til isolering af lymfocytter fra disse rum er nødvendig som antallet af efterforskerne at udforske immun hændelser, som forekommer i tarmen accelererende. Flere forskergrupper har offentliggjort protokoller, der deler flere lignende processer til isolering af immunceller fra musen lille tarm rum3,4,5,6,7 . Der er dog flere tekniske forskelle blandt dem afhængigt af fokus for den enkelte protokol. For eksempel, med fokus på isolere immunceller fra LP, analyserer én protokol konsekvensen af forskellige enzymatiske digestions på cellernes levedygtighed, celle overflade markør udtryk og sammensætningen af isolerede immunceller5. En anden protokol fremhæver en hurtig og reproducerbar metode til isolation af lymfocytter uden tæthed centrifugering6. Endelig findes bestemte protokoller også med henblik på isolering af mononukleære fagocytter fra forskellige væv lag af tyndtarmen7. Her, er en yderst reproducerbare protokol, der giver mulighed for sekventielle isolation af lymfocyt befolkning fra MLN, PP, LP og IEL rum af tyndtarmen præsenteret.
Vi fokuserer på isolere højtrenset befolkninger fra de LP og IEL rum, der er stort set fri for forurenende stoffer fra andre intestinal rum. Dette udbredt meget protokol producerer et højt udbytte af maksimalt ren og levedygtige lymfocytter indenfor acceptabel tid begrænsninger4,8,9,10,11,12. Denne protokol også sikrer isolering af lymfocytter fra LP og IEL rum med minimal afdelingsmæssig krydskontaminering, at en bona fide mulighed for at studere lymfocytter i disse særskilte rum. De isolerede lymfocytter kan underkastes yderligere manipulationer som flow cytometric analyse og funktionalanalyse. Denne protokol har været anvendt med succes til isolering af lymfocytter fra mus tyndtarmen og tyktarmen i løbet af bakterielle infektioner som Listeria monocytogenes, Salmonella typhimuriumog Yersinia pseudotuberculosis infektioner og inflammatoriske tilstande såsom kemiske - og patogen-induceret colitis. Denne protokol kan også bruges til at isolere medfødte immun celler såsom dendritiske celler, makrofager, neutrofile og monocytter fra mus tyndtarmen og tyktarmen.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">HEPES</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:119px;">BP310-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine&#160;</td>
			<td style="width:123px;">Sigma-aldrich</td>
			<td style="width:119px;">G3126-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Penicillin-Streptomycin</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Gentamicin</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:119px;">15710-072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Sodium Hydroxide</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S318-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">RPMI 1640</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:119px;">21870-076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Sodium bicarbonate</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S233-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Fetal bovine serum</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:119px;">26140-079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">10x Hanks&#39; balanced salt solution</td>
			<td style="width:123px;">Sigma-aldrich</td>
			<td style="width:119px;">H4641-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1,4-Dithioerythritol</td>
			<td style="width:123px;">Sigma-aldrich</td>
			<td style="width:119px;">D9680-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">0.5M EDTA, pH 8.0</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:119px;">15575-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calsium chloride hexahydrate</td>
			<td style="width:123px;">Sigma-aldrich</td>
			<td>21108-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride hexahydrate</td>
			<td style="width:123px;">Sigma-aldrich</td>
			<td>M2670-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase, Type I</td>
			<td style="width:123px;">Life Technologies</td>
			<td>17100-017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">DG gradient stock solution (Percoll)&#160;</td>
			<td style="width:123px;">GE Healthcare</td>
			<td style="width:119px;">17-0891-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Red Blood Cell Lysis Buffer</td>
			<td style="width:123px;">Biolegend</td>
			<td style="width:119px;">420301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70-&#181;m cell strainer&#160;</td>
			<td style="width:123px;">Corning</td>
			<td style="width:119px;">352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">14 mL Polypropylene Round-Bottom Tube</td>
			<td style="width:123px;">Corning</td>
			<td style="width:119px;">352059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Erlenmeyer flask&#160;</td>
			<td style="width:123px;">Kimble</td>
			<td style="width:119px;">26500R-50mL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Magnetic stirrer</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:119px;">50094596</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Stir bar</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">14-512-148</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating lymphocytes from the mouse small intestinal immune system

Tarm immunforsvaret spiller en væsentlig rolle i at opretholde den barriere funktion i mave-tarmkanalen ved at generere tolerante svar til kosten antigener og commensal bakterier mens montering effektiv immunrespons til enteropathogenic mikrober. Derudover er det blevet klart, at lokale intestinal immunitet har en dybtgående indvirkning på fjernt og systemisk immunitet. Det er derfor vigtigt at undersøge hvordan en tarm immunrespons er induceret og immunologiske resultatet af reaktionen er. Her, en detaljeret protokol er beskrevet til isolering af lymfocytter fra tyndtarmen induktive sites som de Peyerske plaques gut-associerede lymfoide væv og afdrypning mesenteriallymfeknuderne lymfeknuder og effektor sites som lamina propria og den intestinale epitel. Denne teknik sikrer isolering af et stort antal lymfocytter fra små tarmens væv med optimal renhed og levedygtighed og minimal afdelingsmæssig krydskontaminering inden for acceptable tidspresset. Den tekniske kapacitet til at isolere lymfocytter og andre immunceller fra tarmens væv gør det muligt for forståelsen af immunrespons gastrointestinale infektioner, kræft og inflammatoriske sygdomme.

En skematisk gengivelse af protokollen er afbildet (figur 1). Lymfocytter i tarmslimhinden induktive og effektor websteder er udpræget organiseret. Peyer's patches (PP) og mesenteriallymfeknuderne lymfeknuder (mio.) indeholder lymfocytter i velorganiseret T-celle områder og B-celle follikler, mens den intestinale epitel indeholder lymfocytter, der er mere diffust fordelt. Lamina propria (LP) indeholder både diffust fordelt lymfocytter og lymfocytter indeho...

Watch this Scientific Journal Video about Isolating Lymphocytes from the Mouse Small Intestinal Immune System at JoVE.com

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

Isolere lymfocytter fra mus Small Intestinal immunsystemet

Her beskriver vi en detaljeret protokol til isolering af lymfocytter fra de induktive websteder inklusive de Peyerske plaques gut-associerede lymfoide væv og afdrypning mesenteriallymfeknuderne lymfeknuder, og effektor herunder lamina propria og den intestinale epitel af lille tarm immunsystemet.

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

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