Name: quantifying human monocyte chemotaxis in vitro and murine lymphocyte trafficking in vivo
Uploaded: 2017-10-30T13:00:00
Description: Watch this Scientific Journal Video about Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo at JoVE.com

Dette arbejde blev støttet af Bernard L. Schwartz Program for læge forskere ved The Rockefeller University, Robertson terapeutiske Udviklingsfond på The Rockefeller University og Sackler Center for biomedicin og ernæring forskning på den Rockefeller University.

alle procedurer blev godkendt af Rockefeller University &#39; s institutionelle Animal Care og brug udvalget. Alle dyr har været opstaldet på specifikt patogenfrie betingelser.
 1. In Vitro Chemotaxis <ol><li>kultur THP-1 celler 6 i Roswell Park Memorial Institute 1640 (RPMI 1640) suppleret med 10% føtal bovint serum (FBS) og 4-(2-hydroxyethyl) -1- piperazineethanesulfonic syre (HEPES, 25 mM) (komplet media), opretholde tætheden mellem 0,5-1,0 x 10 <sup...

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	<li>Griffith, J. W., Sokol, C. L., Luster, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+and+chemokine+receptors:+positioning+cells+for+host+defense+and+immunity.">Chemokines and chemokine receptors: positioning cells for host defense and immunity.</a> Annu Rev Immunol. 32, 659-702 (2014).</li><li>Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traffic+signals+for+lymphocyte+recirculation+and+leukocyte+emigration:+the+multistep+paradigm.">Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm.</a> Cell. 76 (2), 301-314 (1994).</li><li>Ponda, M. P., Breslow, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+stimulation+of+CCR7+chemotaxis+due+to+coagulation+factor+XIIa-dependent+production+of+high-molecular-weight+kininogen+domain+5.">Serum stimulation of CCR7 chemotaxis due to coagulation factor XIIa-dependent production of high-molecular-weight kininogen domain 5.</a> Proc Natl Acad Sci U S A. 113 (45), E7059-E7068 (2016).</li><li>Boyden, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemotactic+effect+of+mixtures+of+antibody+and+antigen+on+polymorphonuclear+leucocytes.">The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes.</a> J Exp Med. 115, 453-466 (1962).</li><li>Chen, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boyden+chamber+assay.">Boyden chamber assay.</a> Methods Mol Biol. 294, 15-22 (2005).</li><li>Tsuchiya, S., Yamabe, M., Yamaguchi, Y., Kobayashi, Y., Konno, T., Tada, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+a+human+acute+monocytic+leukemia+cell+line+(THP-1).">Establishment and characterization of a human acute monocytic leukemia cell line (THP-1).</a> Int J Cancer. 26 (2), 171-176 (1980).</li><li>Leonard, E. J., Yoshimura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+monocyte+chemoattractant+protein-1+(MCP-1).">Human monocyte chemoattractant protein-1 (MCP-1).</a> Immunol Today. 11 (3), 97-101 (1990).</li><li>Crouch, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+fluorescence+based+assay+of+neurite+formation.">An automated fluorescence based assay of neurite formation.</a> J Neurosci Methods. 104 (1), 87-91 (2000).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9 (7), 671-675 (2012).</li><li>Poot, M., Kavanagh, T. J., Kang, H. C., Haugland, R. P., Rabinovitch, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+cell+cycle-dependent+changes+in+cell+thiol+level+by+combining+a+new+laser+dye+with+Hoechst+33342.">Flow cytometric analysis of cell cycle-dependent changes in cell thiol level by combining a new laser dye with Hoechst 33342.</a> Cytometry. 12 (2), 184-187 (1991).</li><li>West, C. A., He, C., Su, M., Swanson, S. J., Mentzer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aldehyde+fixation+of+thiol-reactive+fluorescent+cytoplasmic+probes+for+tracking+cell+migration.">Aldehyde fixation of thiol-reactive fluorescent cytoplasmic probes for tracking cell migration.</a> J Histochem Cytochem. 49 (4), 511-518 (2001).</li><li>Kim, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+greater+chemotactic+network+for+lymphocyte+trafficking:+chemokines+and+beyond.">The greater chemotactic network for lymphocyte trafficking: chemokines and beyond.</a> Curr Opin Hematol. 12 (4), 298-304 (2005).</li></ol>

Kvantificering af immun celle migration kan opnås ved hjælp af simple og hurtige undersøgelser både in vitro og i vivo. Vi vise i vitro chemotaxis af humane monocytter som svar på en MCP-1 forløb og brystforstørrelse serum. In vivo, donor murine splenocytes var varierende mærket og efter adoptiv overførsel, blev inddrevet fra modtagere dyr.
Ved hjælp af en Pladelæser har fordel af prøveudtagningssteder i flere tid (så ofte som hver 30 s) og auto...

Robust immunitet kræver komplekse tidsmæssige og rumlige koordinering af et utal af celletyper for at reagere hensigtsmæssigt på skade, infektion og generere self-tolerance. Flere dusin chemokine receptorer og deres tilsvarende ligander er blevet opdaget og karakteriseret giver molekylære mekanismer af hvilke specifikke celler kan rettes til en bestemt væv på et bestemt tidspunkt. Således, at studere chemotaxis og migration er en uundværlig bestanddel af immunologi forskning. Faktisk beskrevet i vitro assay for nylig blev brugt som en screeningsværktøj til at identificere en kemotaktisk cofaktor, der accelererer chemotaxis af T-celler mod C-C kemokiner 19 og 213. Formålet med metoderne beskrevet her er at tillade kvantitative vurderinger af immunforsvar celle chemotaxis in vitro- og in vivo.
Boyden (celle migration og invasion) kammer assay er en billig, reproducerbare og hurtig metode til at vurdere celle migration4,5. I den standard assay, den øverste kammer er seedet med celler, og er adskilt af en porøs indsætte fra en nedre kammer, hvori cellerne vandrer. På det ønskede tidspunkt, kan celler, der har migreret til undersiden af insertet fast og farves til kvantitering af lysmikroskopi. Men sådanne målinger begrænse dataindsamling til et enkelt slutpunkt, som udelukker dynamisk dataindsamling og kan kræve omfattende optimering til at fastslå det optimale tidspunkt for analyse. Her, er flere tilpasninger beskrevet som tillader real-time, kvantitative og multipleksede målinger af chemotaxis i vitro.
For i vivo undersøgelser, bruges en funktionel slutpunkt, nemlig specifik akkumulering af celler i et givet væv rum. Forud mærket donor celler er indført i modtagerens dyr gennem adoptiv overførsel. Disse donor celler kan efterfølgende identificeres ved flowcytometri efter modtagerens væv høst. Også præsenteret er en co mærkning strategi, der giver mulighed for bestemmelse af handel med forskellige celletyper inden for en enkelt modtager dyr. Denne metode fjerner Inter animalske variationen fra celle indsprøjtning, og tegner sig for fysiologisk Inter animalske variabilitet.

<table>
	<tbody>
		<tr>
			<td>RPMI-1640</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ATCC</td>
			<td>30-2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flask</td>
			<td>Corning</td>
			<td>353136</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein AM</td>
			<td>Thermo Fisher Scientific</td>
			<td>C1430</td>
			<td>Excitation: 485nm, Emission: 520nm</td>
		</tr>
		<tr>
			<td>Cell culture dish</td>
			<td>Corning</td>
			<td>1007</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Corning</td>
			<td>353504</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoroblok Fibronectin Insert</td>
			<td>Corning</td>
			<td>CB354597</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>Corning</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Cell Signaling Technology</td>
			<td>9998S</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Recombinant MCP-1</td>
			<td>Peprotech</td>
			<td>300-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Adult bovine Serum</td>
			<td>Sigma</td>
			<td>B9433</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS with calcium and magnesium; no phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>14025-092</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax M2e plate reader</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX71 inverted fluorescence microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isothesia</td>
			<td>Henry Schein animal health</td>
			<td>11695-6776-2</td>
			<td>Isofluorane anesthesia</td>
		</tr>
		<tr>
			<td>Cell strainer 40um nylon</td>
			<td>Falcon Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK lysing buffer</td>
			<td>Quality Biological</td>
			<td>118-156-101</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTracker Orange CMTMR Dye</td>
			<td>Thermo Fisher Scientific</td>
			<td>C2927</td>
			<td>Excitation: 541nm, Emission: 565nm</td>
		</tr>
		<tr>
			<td>CellTracker Green CMFDA Dye</td>
			<td>Thermo Fisher Scientific</td>
			<td>C2925</td>
			<td>Excitation: 485nm, Emission: 520nm</td>
		</tr>
		<tr>
			<td>5ml syringe</td>
			<td>BD Syringe</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>1ml TB syringe</td>
			<td>BD Syringe</td>
			<td>309625</td>
			<td></td>
		</tr>
		<tr>
			<td>THP-1 cell line</td>
			<td>ATCC</td>
			<td>TIB-202</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 421 anti-mouse CD3 Antibody</td>
			<td>Biolegend</td>
			<td>100228</td>
			<td>Excitation: 405nm, Emission: 421nm</td>
		</tr>
		<tr>
			<td>Brilliant Violet 605 anti-mouse CD19 Antibody</td>
			<td>Biolegend</td>
			<td>115540</td>
			<td>Excitation: 405nm, Emission: 603nm</td>
		</tr>
		<tr>
			<td>96-well round bottom plate</td>
			<td>Corning</td>
			<td>353077</td>
			<td></td>
		</tr>
		<tr>
			<td>LSRII</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow cytometer</td>
		</tr>
	</tbody>
</table>

quantifying human monocyte chemotaxis in vitro and murine lymphocyte trafficking in vivo

Chemotaxis er migration langs en særlige kemiske gradient1. Kemokiner er kemotaktisk cytokiner, der fremmer cellulær handel med anatomiske og tidsmæssige specificitet2. Chemotaxis er en kritisk funktion af lymfocytter og andre immunceller, der kan være kvantitativt vurderet in vitro. Dette manuskript beskriver metoder, der foretages evaluering af chemotaxis, både in vitro- og i vivo, for forskellige celletyper, herunder cellelinjer og indfødte celler. In vitro-, plade-baseret format tillader sammenligning af flere betingelser samtidig i realtid, og kan afsluttes inden for 1-4 h. In vitro assay betingelser kan manipuleres til at introducere agonister og antagonister, som godt som differentiere chemotaxis fra chemokinesis, som er tilfældig bevægelse. For i vivo menneskehandel vurderinger, kan immunceller mærket med flere fluorescerende farvestoffer og bruges til adoptiv overførsel. Den differentierede mærkning af celler giver mulighed for blandet cellepopulationer skal føres ind i de samme dyr, dermed faldende varians og reducere antallet af dyr, der er nødvendige for et tilstrækkeligt powered eksperiment. Migration til lymfoid væv forekommer i så lidt som 1 h, og flere tissue rum kan smages. Flowcytometri efter væv høst giver mulighed for en hurtig og kvantitativ analyse af de vandrende mønstre af flere celletyper.

Når du bruger calcein-AM farvestof, vil besigtigelse af celler bekræfte etiket optagelse (figur 1). Automatiseret fluorescerende aflæsninger vil spore migration som celler transit på undersiden af indsatsen over tid. Disse data viser klart en induktion af celle migration mod MCP-1 og en forstærkning af responset ved serum (figur 2A). Afhængigt af styrken af den migrerende stimulus, er der en forsinkelse på mindst 15 min. f...

Watch this Scientific Journal Video about Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo at JoVE.com

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Kvantificering af humane monocyt Chemotaxis In Vitro og Murine lymfocyt handel In Vivo

Protokoller for kvantitativ vurdering af lymfocyt chemotaxis og migration er vigtige værktøjer for immunologi forskning. Her, er en in vitro- protokollen beskrevet, at tilladelser real-time, multipleksede evaluering af celle migration, såvel som en supplerende i vivo teknik muliggør sporing af indfødte celler til milten.

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

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