Name: isolation, characterization, and purification of macrophages from tissues affected by obesity-related inflammation
Uploaded: 2017-04-03T14:00:00
Description: Watch this Scientific Journal Video about Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation at JoVE.com

Nos gustaría dar las gracias a las instalaciones centrales para citometría de flujo en el Pennsylvania estado Universidad Milenio ciencia compleja.

Todos los protocolos experimentales (secciones 1, 1.2 y 1.3) fueron aprobados por el institucional Animal Care y el Comité uso (IACUC) Universidad Estatal de Pensilvania.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>Tejido</td>
			<td>Preparación de Buffers de disociación</td>
			<td>Volumen final</td>
			<td>Almacenamiento de información</td>
		</tr>
<tr>
<td>Tejido adiposo blanco (WAT)</td>
			<td>
Almacenador intermediario d...

<ol>
	<li>Davies, L. C., Taylor, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-resident+macrophages:+Then+and+Now.">Tissue-resident macrophages: Then and Now.</a> Immunology. 144 (4), 541-548 (2015).</li><li>Kahn, S. E., Hull, R. L., Utzschneider, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+linking+obesity+to+insulin+resistance+and+type+2+diabetes.">Mechanisms linking obesity to insulin resistance and type 2 diabetes.</a> Nature. 444 (12), 840-846 (2006).</li><li>Van Gaal, L. F., Mertens, I. L., De Block, C. 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J., Herre, M., Ley, K., Galkina, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+analysis+of+immune+cells+within+murine+aortas.">Flow cytometry analysis of immune cells within murine aortas.</a> J Vis Exp. (53), e2848 (2011).</li><li>Salamone, M., Saladino, S., Pampalone, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Dissociation+and+Primary+Cells+Isolation+Using+Recombinant+Collagenases+Class+I+and+II.">Tissue Dissociation and Primary Cells Isolation Using Recombinant Collagenases Class I and II.</a> Chemical Engineering Transactions. 38, 247-252 (2014).</li><li>Wells, R. 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N., 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+Ron+Receptor+Tyrosine+Kinase+Regulates+Macrophage+Heterogeneity+and+Plays+a+Protective+Role+in+Diet-Induced+Obesity,+Atherosclerosis,+and+Hepatosteatosis.">The Ron Receptor Tyrosine Kinase Regulates Macrophage Heterogeneity and Plays a Protective Role in Diet-Induced Obesity, Atherosclerosis, and Hepatosteatosis.</a> J Immunol. 197 (1), 256-265 (2016).</li><li>Yu, Y. R. A., O'Koren, E. G., 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+protocol+for+the+comprehensive+flow+cytometric+analysis+of+immune+cells+in+normal+and+inflamed+murine+non-lymphoid+tissues.">A protocol for the comprehensive flow cytometric analysis of immune cells in normal and inflamed murine non-lymphoid tissues.</a> PLoS ONE. 11 (3), 1-23 (2016).</li><li>Zizzo, G., Hilliard, B. A., Monestier, M., Cohen, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+clearance+of+early+apoptotic+cells+by+human+macrophages+requires+&amp;#34;M2c&amp;#34;+polarization+and+MerTK+induction.">Efficient clearance of early apoptotic cells by human macrophages requires &amp;#34;M2c&amp;#34; polarization and MerTK induction.</a> J Immunol. 100 (2), 130-134 (2012).</li><li>Jablonski, K. A., Amici, S. 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Modelos de trastorno metabólico inducido por la dieta que imitan comorbilidades tales como ateroesclerosis, esteatosis simple, esteatohepatitis y diabetes tipo 2 se utilizan extensivamente para entender mejor los mecanismos moleculares subyacentes de progresión de la enfermedad. Digestión depende de la colagenasa a menudo se utiliza para disociar los tejidos para liberar las células de la matriz extracelular (ECM)16,27. Enzimas como la colagenasa interrumpen ...

Modelos de ratón se han utilizado para estudiar la dinámica de enfermedades humanas. Correcto aislamiento de las células residentes del tejido de los ratones en un estado enfermo puede proporcionar una plataforma para la comprensión de las contribuciones moleculares y celulares en la patogenia de una enfermedad1. Un trastorno que es de vital importancia es la obesidad. La incidencia de la obesidad sigue aumentando en todo el mundo en paralelo con la resistencia a la insulina y tipo 2 mellitus de la diabetes, enfermedad cardiovascular y enfermedad del hígado graso2,3. Consumo excesivo de nutrientes es más sesgado por disminución de la actividad física provocando señales alteradas provenientes del tejido adiposo, que puede alterar el medio celular de otros tejidos periféricos como el hígado y aorta4. Tal interrupción de la homeostasis metabólica resulta en una inflamación sistémica crónica de bajo grado del5.
Activación clásica del residente a la aorta y el hígado, así como su contratación al tejido adiposo blanco (WAT) de macrófagos ha demostrado no sólo iniciar el dysregulation de señales metabólicas sino también sostener la inflamación6,7. La heterogeneidad fenotípica y funcional de los macrófagos está fuertemente asociada con la patogénesis de la obesidad relacionada con comorbilidades7. La plasticidad dinámica de polarización macrófagos permite para que estas células exhiben una variedad de fenotipos activados que coordinan la progresión y resolución de8de la inflamación. Mientras que clásico activados macrófagos (M1) están implicados en la propagación de la inflamación, macrófagos alternativamente activados (M2) se han asociado con resolución y el tejido de reparación9,10.
Como el cuerpo somete a estrés metabólico, tejido adiposo blanco se acumula anormalmente. El tejido adiposo ampliado atrae y retiene las células inflamatorias que alteran profundamente la función del adipocito normal para promover la resistencia a la insulina, hiperglucemia y diabetes mellitus tipo 2 en última instancia, resistencia a la insulina o hiperglucemia11, 12. En paralelo, remodelaciones de tejido adiposo blanco en respuesta a señales inflamatorias por infiltran clásico activado macrófagos (cajeros automáticos) de tejido adiposo (M1) de13,14. Este órgano multicelular ejerce una cascada de señales que hace descarrilar la función normal de otros órganos del cuerpo tales como la aorta y el hígado4.
El hígado es una potencia metabólica que se adapta en respuesta a estímulos procedentes de cerca dysregulated WAT15. Macrófagos hepáticos o células de Kupffer, en respuesta a cambios metabólicos, secretan citoquinas inflamatorias que transforman tanto parénquima y fenotipo de la célula no parenquimales y promoción la remodelación tisular. Acumulación de lípidos hepáticos, inflamación, depósitos de matriz extracelular excesivo, necrosis y función eventual pérdida sigue los insultos inflamatorios contribuyendo a la amplia gama de daño hepático asociados con enfermedad del hígado graso no alcohólico 1617,de,18.
En paralelo a WAT comprometido y función hepática, arterias grandes acumulan lípidos dentro de la pared arterial como el cuerpo somete a estrés metabólico crónico19. Acumulación lipídica arterial desencadena la secreción de quimioquinas por las células endoteliales activadas y posterior reclutamiento de monocitos20. Una vez reclutados, monocitos proliferan, diferencian, ingieren las lipoproteínas y se convierten en células espumosas. Aterogénesis es iniciada y sostenida por la actividad proinflamatoria de reclutados y macrófagos cargados de lípidos residente de tejido. Sucumbir a las señales de estrés extracelular e intracelular transmitidas en este microambiente aterogénico, estos macrófagos participan luego en una cascada de señalización de apoptosis. Como estas células espumosas mueren, contribuyen a su contenido de lípidos que llena a la base necrótica de la lesión, que entonces conduce a ruptura de placa, infarto de miocardio y accidente cerebrovascular.
Colectivamente, la heterogeneidad de los fenotipos de macrófagos organiza en parte la obesidad inducida por cambios inflamatorios en los tejidos normales como WAT, hígado y aorta8,21. Caracterización de reclutados y macrófagos residentes pueden proporcionar la penetración en posibles dianas moleculares que manipulan macrófago fenotipo1. Para caracterizar eficazmente los macrófagos de los tejidos inflamados inducida por la obesidad, una suspensión unicelular se puede obtener a través de la digestión enzimática. Tales protocolos de disociación deben ser eficaces en degradar suficientemente tejido conectivo mientras que minimiza la muerte celular inmune y proporciona rendimiento óptimo de la célula. La mezcla de la enzima es dependiente en el tipo de tejido y su maquillaje estructural. Tejidos resistentes como la aorta requiere mayor actividad enzimática, en comparación con el hígado y el WAT, para lograr la disociación del tejido. De la suspensión de la célula, macrófagos residentes pueden ser inequívocamente caracterizados o aislados para mayores análisis aguas abajo como perfilamiento transcripcional.
Aquí se describe un protocolo específico de tejido que utiliza tejido dependiente de colagenasa digestión y citometría de flujo policromáticos efectivamente aislar y caracterizar macrófagos residentes de tejido obtenidos de la dieta tradicional inducida por obesidad, ateroesclerosis, esteatosis simple y esteatohepatitis modelos de ratón. Simultánea de marcadores de superficie celular con anticuerpos contra leucocitos (CD45 o CD11b) y macrófagos - antígenos específicos (F4/80) es de uso frecuente para identificar a las poblaciones de macrófagos22. Celular activado por fluorescencia (FACS) de clasificación es una estrategia poderosa que se utiliza para ordenar estas poblaciones identificadas en alta pureza. La población clasificada puede evaluarse entonces perfiles de gen específico fenotipo mediante análisis molecular aguas abajo (por ejemplo, reacción en cadena de polimerasa cuantitativo en tiempo real)23. Aunque citometría de flujo estándar y clasificación de celda basados en citometría de flujo son herramientas poderosas en la distinción de macrófagos dentro de una suspensión sumamente heterogéneos, los protocolos anteriores deben optimizarse para asegurar la salida exitosa. En este estudio, se describen los protocolos que efectivamente aislaran y caracterizan los macrófagos específicos de tejido viable; más importante aún, este estudio proporciona penetración crucial en cuestiones técnicas que a menudo se presentan, así como de estrategias proactivas y solución de problemas para evitar o resolver.

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			<td height="21" style="height:21px;width:321px;">26G x 5/8 in Needles</td>
			<td style="width:151px;">BD</td>
			<td style="width:119px;">305115</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:321px;">23G x 0.75 in needle x 12 in. tubing Blood Collection Set</td>
			<td style="width:151px;">BD</td>
			<td style="width:119px;">367297</td>
			<td style="width:349px;">Used for cannulation of subhepatic IVC during liver perfusion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">21G 1 1/2 in. Needles</td>
			<td style="width:151px;">BD</td>
			<td style="width:119px;">305156</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">1mL syringe with rubber stops</td>
			<td style="width:151px;">BD</td>
			<td style="width:119px;">309659</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">10mL Syringes</td>
			<td style="width:151px;">BD</td>
			<td style="width:119px;">309604</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">1mL Syringe</td>
			<td style="width:151px;">BD</td>
			<td style="width:119px;">309659</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">F4/80 PE</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">123110</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">CD11c PE/Cy7</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">117318</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">CD11b PE/Cy5</td>
			<td style="width:151px;">eBioscience</td>
			<td style="width:119px;">15-0112-81</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Anti-mouse CD16/32 Fc Block&#160;</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">101320</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">CD45 Pacific Blue</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">103126</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">PE Rat IgG2a</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">400508</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">PE/Cy7 Armenian Hamster IgG</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">400922</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">PE/Cy5 Rat IgG2b</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">400610</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Pacific Blue Rat IgG2c</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">400717</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Dulbecco&#8217;s Modified Eagle Medium (DMEM)&#160;</td>
			<td style="width:151px;">Cellgro</td>
			<td style="width:119px;">15-017-CV</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">1X Phosphate Buffered Saline (PBS)</td>
			<td style="width:151px;">Cellgro</td>
			<td style="width:119px;">21-031-CV</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">70 micron cell strainers</td>
			<td style="width:151px;">Corning, Inc.</td>
			<td style="width:119px;">352350</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">&#160;1.7 mL microcentrifuge tube&#160;</td>
			<td style="width:151px;">Denville</td>
			<td style="width:119px;">C2170</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:321px;">Paraformaldehyde Aqueous Solution -16X</td>
			<td style="width:151px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">CAS #30525-89-4</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:321px;">Micro Dissecting Scissors, 3.5 inch, Straight, 23mm, Sharp&#160;</td>
			<td>Stoelting</td>
			<td>52132-10P</td>
			<td style="width:349px;">Used for general dissecting purposes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Forceps, 4in, full curve, 0.8mm</td>
			<td>Stoelting</td>
			<td>52102-37P&#160;</td>
			<td style="width:349px;">Used for general dissecting purposes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Spring dissection scissors &#8211; 3 mm Cutting edge&#160;</td>
			<td style="width:151px;">Fine Science Tool</td>
			<td style="width:119px;">15000-10</td>
			<td style="width:349px;">Used for aorta dissection Steps 1.3.3.17 to 1.3.3.28</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Curved 0.07 x 0.04 mm Tip Forceps&#160;</td>
			<td style="width:151px;">Fine Science Tool</td>
			<td style="width:119px;">11297-10</td>
			<td style="width:349px;">Used for aorta dissection Steps 1.3.3.17 to 1.3.3.28</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Hemostatic Forceps (Curved)</td>
			<td style="width:151px;">Fine Science Tool</td>
			<td style="width:119px;">13021-12</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Heparin Sodium Salt</td>
			<td style="width:151px;">Fischer Scientific</td>
			<td style="width:119px;">9041-08-1</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">35mm Cell Culture/Petri Dishes</td>
			<td style="width:151px;">Fischer Scientific</td>
			<td style="width:119px;">12-565-90</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Polystyrene Petri Dishes (10 cm) w/lid</td>
			<td style="width:151px;">Fischer Scientific</td>
			<td style="width:119px;">08-757-100D</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">15mL Conical Centrifuge Tubes (Polypropylene)</td>
			<td style="width:151px;">Fischer Scientific</td>
			<td style="width:119px;">14-959-53A</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">50mL Conical Centrifuge Tubes&#160;(Polypropylene)</td>
			<td style="width:151px;">Fischer Scientific</td>
			<td style="width:119px;">14-432-22</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">5mL Round-Bottom Polystyrene Tubes&#160;</td>
			<td style="width:151px;">Fischer Scientific</td>
			<td style="width:119px;">14-959-5</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Fetal Bovine Serum</td>
			<td style="width:151px;">Gemini Bio-Products</td>
			<td style="width:119px;">100-106</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:321px;">Ethanol (Stock Ethyl Alcohol Denatured, Anyhydrous)</td>
			<td style="width:151px;">Millipore</td>
			<td style="width:119px;">EX0285-1</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Bovine Serum Albumin</td>
			<td style="width:151px;">Rockland</td>
			<td style="width:119px;">BSA-50</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">HEPES</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Collagenase Type II</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">C6885</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Collagenasse Type XI</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">C7657</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Hyaluronidase Type I</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3506</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">DNAse</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">DN25</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Collagenase Type I</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">C0130</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">NaOH</td>
			<td style="width:151px;">Sigma Aldrich</td>
			<td style="width:119px;">1310-73-2&#160;</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:321px;">CaCl2</td>
			<td style="width:151px;">Sigma Aldrich</td>
			<td style="width:119px;">449709-10G</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">500mL beaker&#160;</td>
			<td style="width:151px;">Sigma Aldrich</td>
			<td style="width:119px;">02-540M</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">4 cm Hemostatic clamp</td>
			<td style="width:151px;">Stoelting</td>
			<td style="width:119px;">52120-40</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Toothed forceps</td>
			<td style="width:151px;">Stoelting</td>
			<td style="width:119px;">52104-33P</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">50 micron Disposable filters</td>
			<td style="width:151px;">Systemex</td>
			<td style="width:119px;">04-0042-2317</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:321px;">Collagenase Type IV</td>
			<td style="width:151px;">ThermoFischer Scientific</td>
			<td style="width:119px;">17104019</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:321px;">Ammonium Chloride Potassium (ACK)</td>
			<td style="width:151px;">ThermoFischer Scientific&#160;</td>
			<td style="width:119px;">A1049201</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Razors (0.22 mm (0.009&#34;))</td>
			<td style="width:151px;">VWR International</td>
			<td style="width:119px;">55411-050</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Texas Red Live/Dead stain</td>
			<td style="width:151px;"></td>
			<td style="width:119px;"></td>
			<td style="width:349px;">Red viability stain (in Figure 1A)</td>
		</tr>
	</tbody>
</table>

isolation, characterization, and purification of macrophages from tissues affected by obesity-related inflammation

La obesidad promueve un estado inflamatorio crónico que es en gran parte mediado por los macrófagos residentes de tejido como macrófagos derivados de monocitos. Obesidad inducida por dieta (DIO) es un modelo valioso en el estudio del papel de la heterogeneidad del macrófago; sin embargo, aislamientos adecuados macrófago son difíciles de adquirir los tejidos inflamados. En este protocolo, describiremos los pasos de aislamiento y pautas necesarias de solución de problemas derivadas de nuestros estudios para la obtención de una adecuada población de macrófagos residentes de tejido de los ratones tras 18 semanas de alto contenido de grasa (HFD) o () alta-grasa/colesterol Intervención de la dieta HFHCD). Este protocolo se centra en tres tejidos de sello estudiados en obesidad y aterosclerosis, incluyendo la aorta, el hígado y tejido adiposo blanco (WAT). Destacamos cómo dualista uso del flujo cytometry puede alcanzar una nueva dimensión de aislamiento y caracterización de los macrófagos residentes de tejido. Una parte fundamental de este protocolo aborda las complejidades subyacentes a digestiones enzimáticas específicas de tejido y macrófagos aislamiento y subsecuente anticuerpo de superficie de la célula de tinción para análisis cytometric del flujo. Este protocolo de direcciones existentes complejidades subyacentes célula fluorescente activada (FACS) de clasificación y presenta aclaraciones a estas complejidades con el fin de obtener la caracterización amplia gama de poblaciones celulares adecuadamente ordenados. Métodos de enriquecimiento alternativo se incluyen para la clasificación de las células, como el hígado denso, lo que permite flexibilidad y manejo de tiempos cuando se trabaja con FACS. En Resumen, este protocolo ayuda al investigador para evaluar la heterogeneidad de macrófago de una multitud de los tejidos inflamados en un determinado estudio y proporciona interesantes consejos para solucionar problemas que han tenido éxito para la caracterización y aislamiento celular favorable de células inmunes en la inflamación mediada por DIO.

Cuando se utiliza la apolipoproteína E deficiente (KO ApoE) C57BL/6 (BL6) ratones mantenidas en una dieta de alta grasa colesterol alto (HCHFD o HCD) para 18 semanas, 1 x 104 - 2 x 104 CD45++ de F4/80 macrófagos aórticos pueden ser aislados cuando las dos muestras son agrupados. Hígado disecado de los ratones alimentados con HFHCD KO ApoE, produjo mayor que 5 x 105 clasificado las células de Kupffer (que depende del tiempo dispon...

Watch this Scientific Journal Video about Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation at JoVE.com

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Este protocolo permite un investigador aislar y caracterizar tejido residente macrófagos en varios sello inflamado los tejidos extraídos de modelos inducida por la dieta de los trastornos metabólicos.

Aislamiento, caracterización y purificación de los macrófagos de los tejidos afectados por la inflamación relacionados con la obesidad

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

The overall goal of this procedure is to effectively isolate and characterize macrophages from tissues commonly affected by diet-induced inflammation such as the liver. This method can help answer key questions in the fields of immunology and inflammation about how tissue-resident macrophage phenotype can dictate the progression of chronic inflammatory diseases. The main advantage of this is that the procedure optimizes the isolation steps needed for obtaining single-cell suspensions derived from tissues impacted by obesity-mediated low-grade chronic inflammation.Demonstrating the procedure will be Joselyn Allen, a graduate student from my laboratory. To isolate the liver, first place an ethanol-soaked mouse in the supine position on a polystyrene foam dissecting board and secure the fore and hind paws. Next, use a medium point tip forceps to grasp the abdominal skin near the urethra opening and use sharp dissecting scissors to create a small incision in the raised skin.Insert the lower blade of the scissors into the incision between the skin and the peritoneum and make a lateral incision from the groin to the chin. Gently pull back the skin to expose the intact peritoneal cavity and thoracic wall and make a lateral incision through the peritoneum. Lift the sternum and carefully incise the diaphragm.Then move the gastrointestinal organs to the side and locate the subhepatic inferior vena cava or IVC. Using hemostatic forceps, clamp the suprahepatic IVC to maintain a localized perfusion and attach a syringe filled with pre-warmed perfusion buffer to a 23 gauge blood collection and infusion set. Gently depress the plunger until the tubing and needle are filled with perfusion buffer and insert the needle parallel to the subhepatic IVC.Proper cannulation of the IVC ensures that the dissociation buffer is locally distributed throughout the liver and peripheral tissues optimizing the tissue digestion. Use a four centimeter hemostatic clamp to secure the needle in place and gently depress the plunger to begin the perfusion. If the needle is appropriately positioned, the color will rapidly flush from the liver and the lobes will become enlarged.Promptly cut the portal vein to allow the perfusion buffer to flow freely through the liver, occasionally gently massaging the lobes between the thumb and forefinger to facilitate the removal of the blood. When only five milliliters of perfusion buffer are left, replace the perfusion buffer syringe with a syringe filled with pre-warmed dissociation buffer, continuing to gently massage the lobes and perfuse the liver until it is fully digested. Confirm a successful digestion by gently pressing the blunt edge of a pair of forceps into the left lateral lobe.An indentation should appear on the surface that slowly fills once the forceps is removed. Then remove the needle and use a short straight blade from a pair of dissecting scissors to carefully cut through the connecting ligaments to excise the whole liver and the gallbladder. Transfer the liver to a 10 centimeter dish containing 10 milliliters of ice cold DMEM and use toothed forceps to grip the severed suprahepatic IVC.Using medium point forceps, apply a rapid stroking motion to each lobe to release the cells from the Glisson's Capsule and pass the saturated DMEM through a 70 micrometer cell strainer in a 50 milliliter conical tube on ice. High yields of viable cells can only be achieved by properly dissociating the liver cells from the Glisson's Capsule. When all of the cells have been collected, invert the tube to gently mix the cell suspension and pellet the cells by centrifugation.Transfer the supernatant to a new 50 milliliter conical tube for three more low-spin centrifugations. After the last centrifugation, transfer the supernatant to a new 50 milliliter tube and centrifuge the cells one more time at a higher speed. Then resuspend the non-parenchymal cell pellet in FACS buffer for counting and dilute the liver cells to the appropriate concentration according to their downstream application in labeled five milliliter round bottom polystyrene tubes.Mice fed a high fat diet or a high fat high cholesterol diet exhibit an increased infiltration of classically activated M1 macrophages in affected tissues such as the aorta. RON receptor expressing aortic CD45 positive F4/80 positive macrophages which demonstrate an anti-inflammatory phenotype are decreased in high fat high cholesterol diet fed mice while pro-inflammatory CD45 positive F4/80 positive CD11c positive macrophages are found in increased numbers in aortas isolated from these animals. Further, sorted RON receptor expressing macrophages derived from digested aortas display an increased expression of Arginase 1 gene, a well-established M2 macrophage marker.Indeed, the characterization of liver-resident macrophages reveals the prevailing phenotype of RON receptor expressing subpopulations with CD11c positive pro-inflammatory macrophage populations demonstrating a decreased expression of genes that are strongly associated with the ant-inflammatory M2 phenotype. Similar trends are observed in the macrophage populations isolated from digested white adipose tissue with pro-inflammatory phenotype macrophage populations exhibiting a decreased surface expression of the RON receptor. Once mastered, this technique can be completed in 20 to 30 minutes per animal if it is performed properly.Following this procedure, other methods such as flow cytometry, fluorescent-activated cell sorting, and/or quantitative PCR can be performed to further characterize the phenotype of the isolated liver-derived tissue-resident macrophages. After watching this video, you should have a good understanding of how to properly isolate tissue-resident macrophages from diseased livers. Thanks for watching and good luck with your experiments.

Research

JoVE Journal

Immunology and Infection

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Aislamiento y caracterización de las células dendríticas y los macrófagos del intestino delgado de ratón

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Investigación

Inmunología e Infección

Proteomic Profiling of Macrophages by 2D Electrophoresis

Proteómico de perfiles de los macrófagos por electroforesis 2D

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Aislamiento de leucocitos a partir de los tejidos murinos en la interfase materno-fetal

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

Un método optimizado para aislar y expandir Invariante Natural Killer células T de ratón Bazo

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

El aislamiento, caracterización y examen funcional de la red celular gingival Inmune

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Caracterización funcional de macrófagos reguladores que inhiben la inmunidad reactiva al injerto

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

Caracterización fenotípica de los macrófagos a partir de riñón de rata por Citometría de Flujo

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Análisis de microglia y macrófagos derivados de monocitos del sistema nervioso central por citometría de flujo

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Aislamiento de Salmonella typhimurium-que contienen fagosomas de los macrófagos

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Aislamiento y caracterización de las vesículas extracelulares de adultos Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...