Name: in vivo photolabeling of cells in the colon to assess migratory potential of hematopoietic cells in neonatal mice
Uploaded: 2018-08-10T14:45:00
Description: Watch this Scientific Journal Video about In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice at JoVE.com

Nitya Jain è stato sostenuto da un premio di transizione di carriera NIH/NIAID 1K22AI116661-01.

Tutte le procedure degli animali sono state effettuate con l'approvazione di e conforme ai istituzionale Animal Care ed uso Committee (IACUC) presso il Massachusetts General Hospital.
Attenzione: Questo protocollo prevede l'utilizzo di un laser di classe 3b (LG3). LG3 laser occhiali di sicurezza devono sempre essere utilizzati quando si opera questo laser. Opportune linee guida formazione e sicurezza devono essere seguite per evitare il rischio di lesioni.
1. progetta...

<ol>
	<li>Maynard, C. L., Elson, C. O., Hatton, R. D., Weaver, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+interactions+of+the+intestinal+microbiota+and+immune+system.">Reciprocal interactions of the intestinal microbiota and immune system.</a> Nature. 489, 231-241 (2012).</li><li>Paun, A., Yau, C., Danska, J. 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+Influence+of+the+Microbiome+on+Type+1+Diabetes.">The Influence of the Microbiome on Type 1 Diabetes.</a> Journal of Immunology. 198, 590-595 (2017).</li><li>Mathis, D., Benoist, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiota+and+autoimmune+disease:+the+hosted+self.">Microbiota and autoimmune disease: the hosted self.</a> Cell Host Microbe. 10, 297-301 (2011).</li><li>Kollmann, T. R., Kampmann, B., Mazmanian, S. K., Marchant, A., Levy, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protecting+the+Newborn+and+Young+Infant+from+Infectious+Diseases:+Lessons+from+Immune+Ontogeny.">Protecting the Newborn and Young Infant from Infectious Diseases: Lessons from Immune Ontogeny.</a> Immunity. 46, 350-363 (2017).</li><li>Jain, N., Walker, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diet+and+host-microbial+crosstalk+in+postnatal+intestinal+immune+homeostasis.">Diet and host-microbial crosstalk in postnatal intestinal immune homeostasis.</a> Nature Reviews in Gastroenterology and Hepatology. 12, 14-25 (2015).</li><li>Mowat, A. M., Agace, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+specialization+within+the+intestinal+immune+system.">Regional specialization within the intestinal immune system.</a> Nature Reviews in Immunology. 14, 667-685 (2014).</li><li>Macpherson, A. J., Uhr, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+protective+IgA+by+intestinal+dendritic+cells+carrying+commensal+bacteria.">Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria.</a> Science. 303, 1662-1665 (2004).</li><li>Mowat, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+basis+of+tolerance+and+immunity+to+intestinal+antigens.">Anatomical basis of tolerance and immunity to intestinal antigens.</a> Nature Reviews in Immunology. 3, 331-341 (2003).</li><li>Diehl, G. 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=Microbiota+restricts+trafficking+of+bacteria+to+mesenteric+lymph+nodes+by+CX(3)CR1(hi)+cells.">Microbiota restricts trafficking of bacteria to mesenteric lymph nodes by CX(3)CR1(hi) cells.</a> Nature. 494 (3), 116-120 (2013).</li><li>Pham, A. H., McCaffery, J. M., Chan, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+lines+with+photo-activatable+mitochondria+to+study+mitochondrial+dynamics.">Mouse lines with photo-activatable mitochondria to study mitochondrial dynamics.</a> Genesis. 50, 833-843 (2012).</li><li>Conway, K. 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=ATG5+regulates+plasma+cell+differentiation.">ATG5 regulates plasma cell differentiation.</a> Autophagy. 9, 528-537 (2013).</li><li>Buzoni-Gatel, D., Lepage, A. C., Dimier-Poisson, I. H., Bout, D. T., Kasper, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+transfer+of+gut+intraepithelial+lymphocytes+protects+against+murine+infection+with+Toxoplasma+gondii.">Adoptive transfer of gut intraepithelial lymphocytes protects against murine infection with Toxoplasma gondii.</a> Journal of Immunology. 158, 5883-5889 (1997).</li><li>Guo, X., Muite, K., Wroblewska, J., Fu, Y. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+Adoptive+Transfer+of+Group+3+Gut+Innate+Lymphoid+Cells.">Purification and Adoptive Transfer of Group 3 Gut Innate Lymphoid Cells.</a> Methods in Molecular Biology. 1422, 189-196 (2016).</li><li>Morton, A. 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=Endoscopic+photoconversion+reveals+unexpectedly+broad+leukocyte+trafficking+to+and+from+the+gut.">Endoscopic photoconversion reveals unexpectedly broad leukocyte trafficking to and from the gut.</a> Proceedings of the National Academy of Sciences of the United States of America. 111, 6696-6701 (2014).</li></ol>

L'identificazione e la caratterizzazione delle cellule che interagiscono e sono influenzati dal microbiota del colon sono importanti e dovrebbero facilitare la comprensione di come le informazioni dal microambiente mucoso viene inoltrati al resto del corpo. Un metodo per studiare la migrazione delle cellule dell'intestino richiede l'isolamento di cellule associato all'intestino, seguita da un trasferimento adottivo in topi destinatari per determinare i modelli di tessuto-homing e funzione12,<...

Il tratto gastrointestinale dei mammiferi ospita centinaia di specie di batteri presenti in un rapporto simbiotico con l'host1. Le cellule immunitarie presenti nell'ambiente locale applicare una coesistenza pacifica con questi microbi e stabilire una barriera protettiva contro le invasioni di agente patogeno. Così, bi-direzionale interazioni tra le cellule immunitarie e il microbiota sono fondamentali per stabilire una comunità commensale che educa il sistema immunitario dell'ospite e imposta la soglia per la reattività immune agli agenti patogeni. Cambiamenti nel microbiche composizione, o disbiosi, può disturbano l'omeostasi immunitaria e perturbano circuiti regolatori che trattengono infiammazioni intestinali che portano a malattie immuno-mediate come diabete di tipo 1 e IBD2,3 .
Il periodo immediatamente dopo la nascita è un'unica finestra inerente allo sviluppo durante il quale le comunità microbiche intestinali cominciano a stabilire allo stesso tempo il sistema immunitario matura4. Il microbiota postnatale non è stabile, con variazioni nella composizione della comunità che si verificano naturalmente e frequentemente5. Le cellule immunitarie che interagiscono con il microbiota risiedono in due distinte posizioni anatomiche nell'intestino - della propria di lamina e l' epitelio intestinale6. Numerosi tipi di cellule immunitarie sono presenti nell'intestino, compreso i linfociti (ad esempio cellule T, cellule B e cellule linfoidi innate) così come le cellule mieloidi (che includono macrofagi, monociti e cellule dendritiche). Queste cellule, anche conosciuto come cellule ematopoietiche, eseguono una moltitudine di funzioni che preservare la barriera intestinale e mantenere l'omeostasi.
Oltre alle loro funzioni di regolamentazione presso siti intestinali, le cellule immuni della mucosa possono anche portare messaggi microbici ai siti extra-intestinali per regolare l'immunità sistemica7,8,9. Questa è un'area di crescente interesse per la ricerca e sottolinea la necessità di metodi per identificare le cellule immunitarie che migrano dai tessuti intestinali al fine di sondare la loro funzione. Il protocollo segnalato qui utilizza un modello di topo commercialmente disponibili in cui viene sfruttata una proteina fluorescente fotoconvertibile alle cellule di etichetta. PhAMasportato topi ubiquistmente esprimono una proteina fluorescente verde di Dendra2 che è irreversibilmente passata a fluorescenza rossa al momento dell'attivazione di raggi ultravioletti (UV) luce10. Utilizzando una cannula di fibra ottica per consegnare 405 nm luce nel colon dei topi neonati, dimostriamo che le cellule ematopoietiche photoconverted, che hanno provenuto o transitato attraverso il colon possono essere trovate nella milza.

<table border="0" cellpadding="0" cellspacing="0" style="width:873px;" width="874">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:873px;">Laser</td>
			<td style="width:201px;"></td>
			<td style="width:167px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:259px;">Light Emitting Diode (LED)</td>
			<td style="width:201px;">THORLABS</td>
			<td style="width:167px;">M405FP1</td>
			<td style="width:247px;">CAUTION: this is a Class 3b laser. Safety goggles must be worn when using the laser. It emits a 405 nm wavelength with a current of 1400 mA. It is fiber-coupled. It accepts SMA connector. https://www.thorlabs.com/thorproduct.cfm?partnumber=M405FP1</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:259px;">LED driver</td>
			<td style="width:201px;">THORLABS</td>
			<td style="width:167px;">LEDD1B</td>
			<td style="width:247px;">Drives a constant current of 1200 mA through the laser. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=2616</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:259px;">Optogenetics patch cable</td>
			<td style="width:201px;">THORLABS</td>
			<td style="width:167px;">M87L01</td>
			<td style="width:247px;">1 m long cable with an SMA connector. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=11405&#38;pn=M87L01#11454</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:259px;">Fiber optic cannula</td>
			<td style="width:201px;">Doric lenses&#160;</td>
			<td style="width:167px;">MFC_480/500-0.5_5mm_ZF1.25_C45</td>
			<td style="width:247px;">5 mm long cannula with an outer diameter of 500 &#181;m and an inner diameter of 480 &#181;m. The NA value is 0.5. The ferrule is zirconia, 1.25 mm OD. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=6036</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">Power supply</td>
			<td style="width:201px;">THORLABS</td>
			<td style="width:167px;">KPS101</td>
			<td style="width:247px;">Supplies 15 V with a current of 2.4 A https://www.thorlabs.com/search/thorsearch.cfm?search=KPS101</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:259px;">LG3 laser safety goggles&#160;</td>
			<td style="width:201px;">THORLABS</td>
			<td style="width:167px;">LG3</td>
			<td style="width:247px;">Orange lenses with 47% visible light transmission https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=762&#38;pn=LG3#2523</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Red light&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>74327-10</td>
			<td style="width:247px;">15 W lamp https://us.vwr.com/store/product/12360027/paterson-safelight-electron-microscopy-sciences</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:873px;">Intestinal cell isolation</td>
			<td style="width:201px;"></td>
			<td style="width:167px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:259px;">Isoflurane</td>
			<td style="width:201px;">Patterson Veterinary</td>
			<td style="width:167px;">07-893-1389</td>
			<td style="width:247px;">CAUTION: inhalation of this anesthetic may cause dizziness, drowsiness, or even unconsciousness. This anesthetic should be used in a Class II hood.&#160; https://www.pattersonvet.com/Supplies/ProductFamilyDetails/PIF_762328?carouselPageNumber=3</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:259px;">1X HBSS</td>
			<td style="width:201px;">Gibco</td>
			<td style="width:167px;">14025076</td>
			<td style="width:247px;">Ca/Mg free https://www.fishersci.com/shop/products/gibco-hbss-calcium-magnesium-no-phenol-red-4/14025076?searchHijack=true&#38;searchTerm=14025076&#38;searchType=RAPID&#38;matchedCatNo=14025076</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Calf Serum</td>
			<td style="width:201px;">Hyclone AZM</td>
			<td style="width:167px;">197696</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:259px;">EDTA</td>
			<td style="width:201px;">Invitrogen</td>
			<td style="width:167px;">15575020</td>
			<td style="width:247px;">0.5 M concentration https://www.thermofisher.com/order/catalog/product/15575020?SID=srch-srp-15575020</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:259px;">DTT</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:167px;">10197777001</td>
			<td style="width:247px;">CAUTION: harmful if swallowed and causes skin irritation. 1 M concentration https://www.sigmaaldrich.com/catalog/product/roche/dttro?lang=en&#38;region=US</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:259px;">HEPES</td>
			<td style="width:201px;">Gibco</td>
			<td style="width:167px;">15630080</td>
			<td style="width:247px;">1 M concentration https://www.thermofisher.com/order/catalog/product/15630080?SID=srch-hj-15630080</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:259px;">Petri dish</td>
			<td style="width:201px;">Corning</td>
			<td style="width:167px;">353004</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/falcon-easy-grip-tissue-culture-dishes-2/08772f?searchHijack=true&#38;searchTerm=08772F&#38;searchType=RAPID&#38;matchedCatNo=08772F</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">70 micron cell strainer</td>
			<td style="width:201px;">Falcon</td>
			<td style="width:167px;">352350</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/falcon-cell-strainers-4/087712</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:259px;">Micro magnetic stir bar</td>
			<td style="width:201px;">Fisherbrand</td>
			<td style="width:167px;">1451364</td>
			<td style="width:247px;">Rinse in 70% ethanol after each use. Rinse several times in distilled water prior to each use. The bar is 8 mm long with an octagonal shape. https://www.fishersci.com/shop/products/fisherbrand-octagonal-magnetic-stir-bars-12/1451364#?keyword=1451364</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:259px;">Magnetic stir plate</td>
			<td style="width:201px;">Corning Laboratory Stirrers</td>
			<td style="width:167px;">440826</td>
			<td style="width:247px;">https://www.coleparmer.com/i/corning-440826-nine-position-stirrer-120-vac-60-hz/8430420?PubID=UX&#38;persist=true&#38;ip=no&#38;gclid=CjwKCAiAqbvTBRAPEiwANEkyCLPLrWABXmOUI0QE53NLV0Owxlcs2V1K6rWbRPOwlcVVDq000FBiQxoCqQAQAvD_BwE</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:259px;">Collagenase</td>
			<td style="width:201px;">Roche</td>
			<td style="width:167px;">5401020001</td>
			<td style="width:247px;">https://www.sigmaaldrich.com/catalog/product/roche/05401020001?lang=en&#38;region=US&#38;gclid=CjwKCAiAjuPRBRBxEiwAeQ2QPhE44qlvxjmo1PYu3zCas3w-_d6P9gKjXW82-c1EOm6NjPHCc5WuixoC_0IQAvD_BwE</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">DNase I</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:167px;">10104159001</td>
			<td style="width:247px;">https://www.sigmaaldrich.com/catalog/product/roche/10104159001?lang=en&#38;region=US</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">1X PBS</td>
			<td style="width:201px;">Gibco</td>
			<td style="width:167px;">20012-027</td>
			<td style="width:247px;">https://www.thermofisher.com/order/catalog/product/20012027?SID=srch-hj-20012-027</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:259px;">Pipet aid</td>
			<td style="width:201px;">Thermo Scientific</td>
			<td style="width:167px;">14387165</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/s1-pipette-fillers/14387165#?keyword=14387165</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">10 mL serological pipet</td>
			<td style="width:201px;">Falcon</td>
			<td style="width:167px;">357530</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/falcon-serological-pipets-bulk-pack-5/p-163659</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">25 mL serological pipet</td>
			<td style="width:201px;">Falcon</td>
			<td style="width:167px;">357515</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/falcon-serological-pipets-bulk-pack-5/p-163659</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">15 mL conical centrifuge tube</td>
			<td style="width:201px;">Thermo Scientific</td>
			<td style="width:167px;">339651</td>
			<td style="width:247px;">https://www.thermofisher.com/order/catalog/product/339650</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">50 mL conical centrifuge tube</td>
			<td style="width:201px;">Thermo Scientific</td>
			<td style="width:167px;">339653</td>
			<td style="width:247px;">https://www.thermofisher.com/order/catalog/product/339650</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:873px;">Single cell suspension</td>
			<td style="width:201px;"></td>
			<td style="width:167px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">Eppendorf tubes</td>
			<td style="width:201px;">Seal-Rite</td>
			<td style="width:167px;">1615-5500</td>
			<td style="width:247px;">Holds 1.5 mL. https://www.usascientific.com/Seal-Rite-1.5-ml-tube.aspx</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:259px;">Tissue homogenizer&#160;</td>
			<td style="width:201px;">Kimble</td>
			<td>K7495400000</td>
			<td style="width:247px;">Requires 2 AA batteries. https://www.fishersci.com/shop/products/kontes-pellet-pestle-cordless-motor-cordless-motor/k7495400000</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:259px;">Homogenizer tips&#160;</td>
			<td style="width:201px;">Kimble</td>
			<td style="width:167px;">7495210590</td>
			<td style="width:247px;">Plastic, 0.5 mL tips https://www.fishersci.com/shop/products/kimble-chase-kontes-pellet-pestle-14/k7495210590#?keyword=7495210590</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">ACK lysing buffer</td>
			<td style="width:201px;">Gibco</td>
			<td style="width:167px;">A10492-01</td>
			<td style="width:247px;">https://www.thermofisher.com/order/catalog/product/A1049201?SID=srch-hj-A10492-01</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">40 micron cell strainer</td>
			<td style="width:201px;">Falcon</td>
			<td style="width:167px;">08-771-1</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/falcon-cell-strainers-4/087711</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:873px;">Antibodies</td>
			<td style="width:201px;"></td>
			<td style="width:167px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:259px;">BV786 anti-mouse CD45&#160;</td>
			<td style="width:201px;">BD</td>
			<td style="width:167px;">564225</td>
			<td style="width:247px;">Clone 3O-F11 https://www.bdbiosciences.com/us/reagents/research/antibodies-buffers/immunology-reagents/anti-mouse-antibodies/cell-surface-antigens/bv786-rat-anti-mouse-cd45-30-f11/p/564225</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Live/Dead</td>
			<td>Invitrogen</td>
			<td>L34962</td>
			<td style="width:247px;">https://www.thermofisher.com/order/catalog/product/L34962</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Other</td>
			<td style="width:201px;"></td>
			<td style="width:167px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Razor blades</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td style="width:247px;">Use for decapitation. https://us.vwr.com/store/product/4548306/vwr-razor-blades</td>
		</tr>
	</tbody>
</table>

in vivo photolabeling of cells in the colon to assess migratory potential of hematopoietic cells in neonatal mice

Comunità batteriche enteriche sono stabilite all'inizio della vita e influenzano la funzione e lo sviluppo delle cellule immuni. Il microbiota neonatale è suscettibile di numerose influenze esterne, tra cui l'uso di antibiotici e dieta, che urta la suscettibilità alle malattie autoimmuni ed infiammatorie. Disturbi come la malattia di viscere infiammatoria (IBD) sono caratterizzati da un massiccio afflusso di cellule immuni agli intestini. Tuttavia, le cellule immunitarie condizionate dal microbiota possono inoltre emigrare fuori gli intestini per influenzare le risposte immunitarie a siti extra-intestinali. Così, c'è una necessità di identificare e caratterizzare le cellule che possono trasportare messaggi microbici dall'intestino ai luoghi distali. Qui, descriviamo un metodo alle cellule di etichetta nel colon dei topi neonati in vivo che consenta la loro identificazione presso siti extra-intestinali dopo la migrazione.

Un cavo in fibra ottica è stato utilizzato per trasportare 405 nm luce in due punti dei topi diasportato 2 - giorno-vecchi PhAM. In precedenti esperimenti, un 30 s esposizione era deciso a dare una massima fotoconversione delle cellule del colon con minima citotossicità (Figura 1A). Di conseguenza, sequenziale 30 esposizioni di s dei diversi segmenti del colon sono state effettuate come descritto nel protocollo. In seguito l'espo...

Watch this Scientific Journal Video about In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice at JoVE.com

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

Il protocollo descritto qui utilizza un approccio di photolabeling in topi neonati per identificare in modo specifico le cellule immuni che emigrano dal colon in siti extra-intestinali. Questa strategia sarà utile per studiare le interazioni ospite-microbiome nella vita in anticipo.

In Vivo Photolabeling delle cellule del colon per valutare il potenziale migratorio delle cellule ematopoietiche in topi neonatali

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to ...

Research

JoVE Journal

Medicine

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

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There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

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Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

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In Vivo Imaging e tracciamento di tecnezio-99m etichetta cellule staminali mesenchimali del midollo osseo in Equine Tendinopatia

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

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Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

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Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

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The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

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Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...