Name: real-time imaging of myeloid cells dynamics in apcmin/+ intestinal tumors by spinning disk confocal microscopy
Uploaded: 2014-10-06T14:00:00
Description: Watch this Scientific Journal Video about Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy at JoVE.com

Vi vil gerne takke Ying Yu for APC MIN / + mus genotypebestemmelse. Denne undersøgelse blev støttet af midler fra INSERM og tilskud (CA057621 og AI053194) fra National Institutes of Health.

BEMÆRK: Alle dyreforsøg blev udført i overensstemmelse med de procedurer, der er godkendt af Institutional Animal Care og brug Udvalg (IACUC), UCSF. Alle de billeddannende eksperimenter var ikke-overlevelse procedurer og dyrene blev aflivet umiddelbart efter afslutningen af ​​billedet erhvervelsen. 1. Generering af mus BEMÆRK: Apc Min / + mus, der bærer en mutation på APC-genet, udvikler spontant 50-100 adenomer i tyndtarmen. ...

<ol>
	<li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+the+next+generation.">Hallmarks of cancer: the next generation.</a> Cell. 144 (5), 646-674 (2011).</li><li>Hanahan, D., Coussens, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accessories+to+the+crime:+functions+of+cells+recruited+to+the+tumor+microenvironment.">Accessories to the crime: functions of cells recruited to the tumor microenvironment.</a> Cancer Cell. 21 (3), 309-322 (2012).</li><li>Schouppe, E., De Baetselier, P., Van Ginderachter, J. A., Sarukhan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instruction+of+myeloid+cells+by+the+tumor+microenvironment:+Open+questions+on+the+dynamics+and+plasticity+of+different+tumor-associated+myeloid+cell+populations.">Instruction of myeloid cells by the tumor microenvironment: Open questions on the dynamics and plasticity of different tumor-associated myeloid cell populations.</a> Oncoimmunology. 1 (7), 1135-1145 (2012).</li><li>Egeblad, M., Nakasone, E. S., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumors+as+organs:+complex+tissues+that+interface+with+the+entire+organism.">Tumors as organs: complex tissues that interface with the entire organism.</a> Dev Cell. 18 (6), 884-901 (2010).</li><li>Lohela, M., Werb, Z. <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+stromal+cell+dynamics+in+tumors.">Intravital imaging of stromal cell dynamics in tumors.</a> Curr Opin Genet Dev. 20 (1), 72-78 (2010).</li><li>Egeblad, 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=Visualizing+stromal+cell+dynamics+in+different+tumor+microenvironments+by+spinning+disk+confocal+microscopy.">Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy.</a> Dis Model Mech. 1 (2-3), 155-167 (2008).</li><li>Ewald, A. J., Werb, Z., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+long-term+in+vivo+imaging+of+tumor-stroma+interactions+in+mouse+models+of+breast+cancer+using+spinning-disk+confocal+microscopy.">Dynamic long-term in vivo imaging of tumor-stroma interactions in mouse models of breast cancer using spinning-disk confocal microscopy.</a> Cold Spring Harb Protoc. 2011 (2), (2011).</li><li>Ewald, A. J., Werb, Z., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+vital+signs+for+long-term+survival+of+mice+under+anesthesia.">Monitoring of vital signs for long-term survival of mice under anesthesia.</a> Cold Spring Harb Protoc. 2011 (2), (2011).</li><li>Ewald, A. J., Werb, Z., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+mice+for+long-term+intravital+imaging+of+the+mammary+gland.">Preparation of mice for long-term intravital imaging of the mammary gland.</a> Cold Spring Harb Protoc. 2011 (2), (2011).</li><li>Nakasone, E. S., Askautrud, H. A., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+drug+responses+in+the+tumor+microenvironment+in+mouse+models+of+breast+cancer.">Live imaging of drug responses in the tumor microenvironment in mouse models of breast cancer.</a> J Vis Exp. (73), e50088 (2013).</li><li>Nakasone, E. 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=Imaging+tumor-stroma+interactions+during+chemotherapy+reveals+contributions+of+the+microenvironment+to+resistance.">Imaging tumor-stroma interactions during chemotherapy reveals contributions of the microenvironment to resistance.</a> Cancer Cell. 21 (4), 488-503 (2012).</li><li>Walther, 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=Genetic+prognostic+and+predictive+markers+in+colorectal+cancer.">Genetic prognostic and predictive markers in colorectal cancer.</a> Nat Rev Cancer. 9 (7), 489-499 (2009).</li><li>Fearon, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+genetics+of+colorectal+cancer.">Molecular genetics of colorectal cancer.</a> Annu Rev Pathol. 6, 479-507 (2011).</li><li>Moser, A. R., Pitot, H. C., Dove, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dominant+mutation+that+predisposes+to+multiple+intestinal+neoplasia+in+the+mouse.">A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse.</a> Science. 247 (4940), 322-324 (1990).</li><li>Su, L. K., 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=Multiple+intestinal+neoplasia+caused+by+a+mutation+in+the+murine+homolog+of+the+APC+gene.">Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene.</a> Science. 256 (5057), 668-670 (1992).</li><li>Watson, A. 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=Epithelial+barrier+function+in+vivo+is+sustained+despite+gaps+in+epithelial+layers.">Epithelial barrier function in vivo is sustained despite gaps in epithelial layers.</a> Gastroenterology. 129 (3), 902-912 (2005).</li><li>McDole, J. 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=Goblet+cells+deliver+luminal+antigen+to+CD103++dendritic+cells+in+the+small+intestine.">Goblet cells deliver luminal antigen to CD103+ dendritic cells in the small intestine.</a> Nature. 483 (7389), 345-349 (2012).</li><li>Xu, C., Shen, Y., Littman, D. R., Dustin, M. L., Velazquez, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+mucosal+homeostasis+via+single-+and+multiphoton+intravital+fluorescence+microscopy.">Visualization of mucosal homeostasis via single- and multiphoton intravital fluorescence microscopy.</a> J Leukoc Biol. 92 (3), 413-419 (2012).</li><li>Haigis, K. 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=Differential+effects+of+oncogenic+K-Ras+and+N-Ras+on+proliferation,+differentiation+and+tumor+progression+in+the+colon.">Differential effects of oncogenic K-Ras and N-Ras on proliferation, differentiation and tumor progression in the colon.</a> Nat Genet. 40 (5), 600-608 (2008).</li><li>Sansom, O. 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=Loss+of+Apc+allows+phenotypic+manifestation+of+the+transforming+properties+of+an+endogenous+K-ras+oncogene+in+vivo.">Loss of Apc allows phenotypic manifestation of the transforming properties of an endogenous K-ras oncogene in vivo.</a> Proc Natl Acad Sci U S A. 103 (38), 14122-14127 (2006).</li><li>Janssen, K. P., 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=APC+and+oncogenic+KRAS+are+synergistic+in+enhancing+Wnt+signaling+in+intestinal+tumor+formation+and+progression.">APC and oncogenic KRAS are synergistic in enhancing Wnt signaling in intestinal tumor formation and progression.</a> Gastroenterology. 131 (4), 1096-1109 (2006).</li><li>Takaku, K., 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=Intestinal+tumorigenesis+in+compound+mutant+mice+of+both+Dpc4+(Smad4)+and+Apc+genes.">Intestinal tumorigenesis in compound mutant mice of both Dpc4 (Smad4) and Apc genes.</a> Cell. 92 (5), 645-656 (1998).</li></ol>

I dette papir, er en detaljeret protokol beskrevet for spinning disk konfokal billeddannelse af myeloide celle dynamik i tarm tumorer i flere timer i et levende dyr, afbildet fra serøse side af tarmen. For at undgå betændelse og at have optimale fysiologiske betingelser, billeddannelse af tarmen skal ske på den intakte organ. Billeddannelse fra serøse side af tarmen er imidlertid en udfordring, fordi lyset skal gå gennem forskellige vævslag såsom glat muskel, før de når epitel. Fok...

Overvældende beviser viser nu, tumormikromiljøet bestående af heterogene cellepopulationer, herunder fibroblaster, endotelceller, immune og inflammatoriske celler, ekstracellulære matrix, og opløselige faktorer spiller en afgørende rolle i initiering og progression af faste tumorer ved at bidrage til næsten alle kendetegnende for kræft 1. Faktisk under tumorprogression, der konstant dynamiske interaktioner mellem transformerede cancerceller og stromale celler, der udvikler sig til at frembringe et gunstigt malignitet 2 mikromiljø. Blandt de immunceller der infiltrerer tumormikromiljøet, myeloide celler er den mest rigelige 3. Bestående af tumorassocierede makrofager (TAM), myeloide afledt suppressor celler (MDSCs), dendritiske celler (DC) og neutrofiler (PMN&#39;er) er myeloide celler rekrutteret fra knoglemarv og gradvist infiltrere tumorer frigive cytokiner, vækstfaktorer og proteaser, som kan fremmetumorvækst og spredning 4. Krydstale mellem cancerceller og myeloide celler er kompliceret, men dynamisk. Således forståelse af karakteren af ​​deres samspil er afgørende for, hvorfor disse celler fremmer udviklingen af ​​kræft i stedet for at deltage i et immunrespons anti-tumor, og kan hjælpe med at finde nye mål at kontrollere den. Direkte observation af intravital mikroskopi giver oplysninger om celle dynamik inden for væv af levende mus 5. En fire-farve, multi-region, mikro-lensed roterende skive konfokalt system blev udformet til at studere stromale celler i brysttumorer 6. Denne tilgang gør det muligt langsigtet kontinuerlig billeddannelse og omfatter flere fordele såsom (a) hurtige billeder erhvervelse at minimere bevægelsesartefakter, (b), langvarig anæstesi, (c) fire farver erhvervelse at følge forskellige celletyper, (d) fluorescensmærkning forskellige tumorale komponenter og (e), observation af forskellige tumor mikromiljøer within samme mus for at undgå mus variabiliteten mus 7-9. Med denne teknologi, har forskellige celletyper adfærd blevet rapporteret i den brysttumorvirus (MMTV) promotordrevet polyom midten T onkogen (PyMT) model, der viser progressive stadier af tumorigenese. Regulatoriske T-lymfocytter (tregs, visualiseret ved den Foxp3 EGFP transgen) vandrer fortrinsvis i nærheden af blodkar mens DC&#39;er (CD11c-DTR-EGFP), carcinoma-associerede fibroblaster (Fsp1 + / + -EGFP) og myeloide celler (c-fms -EGFP) udviser højere motilitet ved tumoren periferien end i tumoren masse. I den tilstand af akut systemisk hypoxi, cellerne er migreret anderledes: tregs stoppe migrere i modsætning til myeloide celler, der fortsætter med at bevæge 6. Desuden, i den samme musemodel, er det blevet vist, at doxorubicin følsomhed ændringer med tumor stadium er lægemiddelfordeling relateret til lægemiddel respons, og doxorubicinbehandling fører til CCR2-afhængig rekruttering af myeloide celler til tumorer. Således kan den direkte billedvisning også bruges til at få indblik i narkotika reaktioner in situ og biologi kemoterapiresistens 10,11. Adenomatøs polypose coli (APC) genmutationer almindeligt forekommende i humane kolorektale adenomer og karcinomer 12 og mutation af en enkelt kopi af APC-genet resulterer i familiær adenomatøs polypose (FAP), som giver en ekstremt høj risiko for tyktarmskræft 13. Den musestamme Apc Min / + bærer en trunkering mutation ved kodon 850 af APC-genet og spontant udvikler multiple intestinale adenomer hele tyndtarmen 14.-16. Langsigtet intravitalt billeddannelse af tarmen er udfordrende på grund af invasiv procedure, idet åbning af bughulen er nødvendig for adgang til tarmen. Kortsigtet direkte billedvisning studies har tidligere været udgivet på sund tarm 17,18, men på lang sigt direkte observation af intestinale tumorer er ikke blevet rapporteret. Et kirurgisk indgreb er designet og raffineres for at visualisere tumorer gennem den serøse overflade af tarmen ved hjælp af intravital roterende skive mikroskopi systemet tidligere er anvendt til billedet brysttumorer 6,10. I dette papir, er en protokol, der er beskrevet, der tillader en at følge adfærden af myeloide celler i tumorer i tyndtarmen ved hjælp af APC Min / + mus.

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	<tbody>
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			<td height="42" style="height:42px;width:216px;"><a name="RANGE!A1:D30">Name of Material/ Equipment</a></td>
			<td style="width:107px;">Company</td>
			<td style="width:141px;">Catalog Number</td>
			<td style="width:227px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ApcMin/+ mice</td>
			<td style="width:107px;">Jackson Laboratory</td>
			<td>2020</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ACTB-ECFP mice</td>
			<td style="width:107px;">Jackson Laboratory</td>
			<td style="width:141px;">3773</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">cfms-EGFP mice</td>
			<td style="width:107px;">Jackson Laboratory</td>
			<td align="right">18549</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2,000 kDa Dextran, rhodamine-conjugated</td>
			<td style="width:107px;">Invitrogen</td>
			<td style="width:141px;">D7139</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Isoflurane</td>
			<td style="width:107px;">Butler Animal Health Supply</td>
			<td style="width:141px;">29450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nitrogen</td>
			<td style="width:107px;">UCSF</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oxygen</td>
			<td style="width:107px;">UCSF</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1X PBS</td>
			<td style="width:107px;">UCSF cell culture facility</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Saline Buffer</td>
			<td style="width:107px;">UCSF cell culture facility</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti-mouse Ly-6G (GR1) antibody AF647</td>
			<td style="width:107px;">UCSF Monoclonal antibody core</td>
			<td style="width:141px;"></td>
			<td>Stock 1mg/ml. Use at 7ug/mouse</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Atropine</td>
			<td style="width:107px;">LARC UCSF</td>
			<td style="width:141px;"></td>
			<td>Use at 1mg/Kg mouse</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alcohol wipes</td>
			<td style="width:107px;">Becton Dickinson</td>
			<td style="width:141px;">326895</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">28G1/2 insulin syringe</td>
			<td style="width:107px;">Becton Dickinson</td>
			<td style="width:141px;">329465</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Remium cover glass</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:141px;">12-548-5M</td>
			<td>24x50-1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Superfrost plus microscope slides</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:141px;">12-550-15</td>
			<td>25x75x1mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Krazy glue</td>
			<td style="width:107px;">Office Max</td>
			<td align="right">7111555</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Betadine</td>
			<td style="width:107px;">LARC UCSF</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Heat blanket</td>
			<td style="width:107px;">Gaymar Industries</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hot bead sterilizer</td>
			<td style="width:107px;">Fine Science Tools</td>
			<td style="width:141px;">18000-45</td>
			<td>Turn ON 30min before use</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Cotton tipped apllicators 6-inch</td>
			<td style="width:107px;">Electron Microscopy Sciences</td>
			<td style="width:141px;">72310-10</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anesthesia system</td>
			<td style="width:107px;">Summit Anesthesia Support</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inverted microscope</td>
			<td style="width:107px;">Carl Zeiss Inc</td>
			<td style="width:141px;">Zeiss Axiovert 200M</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">stage insert</td>
			<td style="width:107px;">Applied Sientific Instrumentation</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mouse Ox oximeter, software and sensors</td>
			<td style="width:107px;">Starr Life Sciences</td>
			<td style="width:141px;">MouseOx</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Nebulizer</td>
			<td style="width:107px;">Summit Anesthesia Support</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Imaris</td>
			<td style="width:107px;">Bitplane</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mManager</td>
			<td style="width:107px;">Vale lab, UCSF</td>
			<td style="width:141px;"></td>
			<td>Open-source software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ICCD camera</td>
			<td style="width:107px;">Stanford Photonics</td>
			<td style="width:141px;">XR-Mega-10EX S-30</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spinning disk confocal sacan-head</td>
			<td style="width:107px;">Yokogawa Corporation</td>
			<td style="width:141px;">CSU-10b</td>
			<td></td>
		</tr>
	</tbody>
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real-time imaging of myeloid cells dynamics in apcmin/+ intestinal tumors by spinning disk confocal microscopy

Myeloide celler er de mest rigelige immunceller i tumorer og har vist sig at fremme tumorudvikling. Moderne intravital billeddiagnostiske teknikker muliggøre observation af levende cellulær adfærd inde i orglet, men kan være en udfordring i nogle typer af kræft som følge af orgel og tumor tilgængelighed såsom tarmen. Direkte observation af intestinale tumorer er ikke tidligere blevet rapporteret. En kirurgisk procedure, der er beskrevet her tillader direkte observation af myeloide celle dynamik inden for de intestinale tumorer i levende mus ved hjælp af transgene fluorescerende reporter mus og injicerbare sporstoffer eller antistoffer. Til dette formål har en fire-farve, multi-region, mikro-lensed roterende skive konfokalt mikroskop, der giver langsigtet kontinuerlig billeddannelse med køb hurtig billede blevet brugt. APC Min / + mus, der udvikler flere adenomer i tyndtarmen krydses med c-fms-EGFP mus for at visualisere myeloide celler og med ACTB-ECFP musat visualisere intestinale epitelceller i krypterne. Procedurer for mærkning forskellige tumor komponenter, såsom blodkar og neutrofiler, samt proceduren for placering af tumor til billeddannelse gennem serøse overflade er også beskrevet. Time-lapse film indsamlet fra flere timers billeddannelse tillader analyse af myeloid celle adfærd in situ i tarmen mikromiljø.

Ved hjælp af spinning disk konfokal mikroskopi, ikke-tumorale og tumorvæv i tyndtarmen af Apc Min / +; ACTB-ECFP, c-fms-ECFP mus kan visualiseres fra den serøse overflade. Efter billedbehandling, er kamera software, der bruges til at analysere og justere købet (supplerende figur 1). Efter intravenøs (iv) injektion af fluorescerende 2.000 kDa dextran-rhodamin og Ly-6G 647 konjugeret antistof, blodkar og PMN&#39;er kan detekteres (figur 1). Peyerpletter d...

Watch this Scientific Journal Video about Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy at JoVE.com

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Real-time Imaging af myeloide celler dynamik i<em&gt; Apc<sup&gt; Min / +</sup</em&gt; Intestinal Tumorer ved Spinning Disk Konfokal mikroskopi

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

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JoVE Journal

Immunology and Infection

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

Intravital Mikroskopi af milten: Kvantitativ analyse af Parasite mobilitet og Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

En TIRF Mikroskopi Technique for i realtid, Samtidig Billeddannelse af TCR og dens associerede signalproteiner

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Påvisning af MicroRNA i Mikroglia af Real-time PCR i Normal CNS og under neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Real-time Live Imaging af T-celle signalering Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

Vurdere antisvampeaktivitet Isoleret alveolære makrofager ved konfokal mikroskopi

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

Real-time Imaging af endotelcellefremkaldt celle vejkryds Under Neutrofil Transmigration under fysiologiske Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

Den mesenterielymfeknuderne Duct kanyle Rat Model: Ansøgning om vurdering af intestinal lymfatisk Drug Transport

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induktion af murine tarmbetændelse ved adoptiv overførsel af effektor CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Høj</sup&gt; T celler i immunsvækkede mus

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Analyse af<em&gt; Yersinia enterocolitica</em&gt; Effector Translokation i værtsceller Brug Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Imaging neutrofiler og monocytter i Mesenterial Veins af Intravital Mikroskopi på bedøvede mus i Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...