Name: isolation of cortical microglia with preserved immunophenotype and functionality from murine neonates
Uploaded: 2014-01-30T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates at JoVE.com

Este trabajo fue apoyado por el NIEHS R01ES014470 (KMZ).

Antes de iniciar este protocolo, recoger ratones recién nacidos en los días postnatales 1-3 (P1-3) en un recipiente estéril anidado con ropa de cama caja original para protegerlo y abrigarlo. Es importante trabajar de manera rápida y eficiente a través de este protocolo para optimizar el rendimiento de la microglia. Por favor consulte la Tabla 1 para la lista completa de reactivos. 1. Elaboración de instrumentos, medios de cultivo, y platos <ol><li> Preparar 10 ml / ...

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Neurochem. 80, 697-705 (2002).</li><li>Uesugi, M., Nakajima, K., Tohyama, Y., Kohsaka, S., Kurihara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonparticipation+of+nuclear+factor+kappa+B+(NFkappaB)+in+the+signaling+cascade+of+c-JunN-terminal+kinase+(JNK)-+and+p38+mitogen-activated+protein+kinase+(p38MAPK)-dependent+tumor+necrosis+factor+alpha+(TNFalpha)+induction+in+lipopolysaccharide+(LPS)-stimulated+microglia.">Nonparticipation of nuclear factor kappa B (NFkappaB) in the signaling cascade of c-JunN-terminal kinase (JNK)- and p38 mitogen-activated protein kinase (p38MAPK)-dependent tumor necrosis factor alpha (TNFalpha) induction in lipopolysaccharide (LPS)-stimulated microglia.</a> Brain Res. , 1073-1074 (2006).</li><li>Beraud, D., 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=Microglial+Activation+and+Antioxidant+Responses+Induced+by+the+Parkinson's+Disease+Protein+alpha-Synuclein.">Microglial Activation and Antioxidant Responses Induced by the Parkinson's Disease Protein alpha-Synuclein.</a> J. 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El presente procedimiento ofrece un método eficaz para el aislamiento de la microglia corticales de ratones neonatales. Este procedimiento tiene un doble beneficio de 1) preservar inmunofenotipo la microglia y la funcionalidad, según lo determinado por proyección de imagen fluorescente, inmunocitoquímica, y ELISA, y, 2) que permite la microglía a madurar en presencia de otras células gliales (astrocitos y oligodentrocytes) antes de la aislamiento, lo que puede facilitar importantes interacciones célula-célula du...

Las células microgliales, los macrófagos de vigilancia de la parénquima del SNC, comprenden aproximadamente el 12% de la población total de células del cerebro de los mamíferos adultos. La microglía se derivan de saco vitelino mieloides precursoras células y varían en densidad celular y la morfología en diferentes regiones cytoarchitectural en el SNC adulto 1-5. En un cerebro adulto sano, la microglia son células pequeñas, ramificadas o polares, con procesos dinámicos finos. En contraste con la morfología de los macrófagos periféricos, microglia demuestran un reposo, de bajo perfil fenotipo en cerebros sanos que pueden aparecer como la inactividad celular, sin embargo, en los estudios de imagen in vivo demuestran que los procesos microgliales se extienden de forma dinámica y se retraen para monitorear su microambiente de una manera que recuerda a &quot; muestreo y de estudio &quot;6,7. La microglía son altamente y diferencialmente sensibles a alteraciones ambientales y fisiopatológicos en el cerebro, de conmutacióndesde un surveillant a un estados considerados como su reposo y activadas comúnmente estatales efectoras, respectivamente. Este interruptor de activación puede estar mediada por la participación de los receptores de membrana de reconocimiento de patrones (PRRS), como los receptores tipo Toll (TLR), que responden a patrones moleculares asociados a patógenos (PAMP), lipoproteínas de saber bacterianas-y derivados-viral , ácidos nucleicos, hidratos de carbono y 8-11. Además de PAMP, parentales también se han mostrado para inducir la activación microglial contra moléculas estériles, no patógenas conocidas como patrones moleculares peligro / daño-asociado (apaga), que representan una perturbación en la homeostasis del SNC, tales como daño celular 12-16. Una vez enganchado, PRRS inician una cascada de señalización intracelular que resulta en cambios en la morfología celular microglial y la expresión génica; específicamente, microglia activada adaptar un fenotipo-ameboide como, translocate la p65 de NF-kappa B subunidad (p65) para el núcleo de la célula, y la upregulate producción y la secreción de citocinas proinflamatorias, tales como factor de necrosis tumoral-α (TNF-α), la interleucina-1 β (IL-1β), junto con especies de oxígeno reactivas (ROS) 16-24. Aunque integral en la respuesta inmune innata del sistema nervioso central, también se han encontrado estas moléculas secretadas para aumentar el estrés oxidativo neuronal, induciendo de ese modo y exacerbando la neurodegeneración en estados de enfermedad tales como Parkinson y la enfermedad de Alzheimer 25-29. Sin embargo, los mecanismos de activación microglial en estados patológicos no se entienden completamente. Por lo tanto, el aislamiento de microglia es una poderosa herramienta de investigación en estos procesos biológicos, como muchos en funciones in vivo de la activación microglial puede ser recapitulado en la cultura. Varios métodos están disponibles para el aislamiento de la microglía, incluyendo el aislamiento a través de un gradiente de Percoll después de la digestión enzimática de tejido del SNC 30,31. Sin embargo, la digestión enzimática puede alterarel inmunofenotipo de las células mediante la reducción de la superficie celular la expresión del antígeno 32, y los resultados en el rendimiento de células por animal inferior que el método descrito en el presente documento. En concreto, se presenta un rendimiento promedio microglia por corteza cachorro de 7,5 x 10 5 células, mientras que los métodos de aislamiento de todo el SNC ya se ha informado a través de un rendimiento gradiente de Percoll 3-5 x 10 5 células 30,33,34. El presente procedimiento evita el uso de enzimas de digestión mediante el aislamiento de la microglia en base a sus propiedades de baja adherencia, preservando así el inmunofenotipo y la funcionalidad de la microglia. En el presente estudio, se describe el aislamiento de las células microgliales de mezcla de culturas gliales derivadas de heterocigotos CX3CR1-GFP (CX3CR1-GFP + / -) neonatales, y C57BL / 6 corticales murinas mediante agitación mecánica en un agitador rotatorio, una extensión de anterioridad publicado 24,35 método. Utilizamos la antigua cepa de ratón para facilitar la visualización de la microglia, comoestos ratones expresan GFP bajo el control del locus endógeno CX3CR1 - un promotor específico de los monocitos 36-38. Este método produce microglial culturas altamente puro con un inmunofenotipo conservados ex vivo como se demuestra por cambios morfológicos, la translocación nuclear de p65, y la secreción de TNF-α cuando son desafiados con lipopolisacárido bacteriano (LPS) o Pam 3 CSK 4, TLR4 y TLR1 / 2 agonistas , respectivamente.

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			<td height="160" style="height:160px;width:233px;">Glucose</td>
			<td style="width:149px;">Sigma</td>
			<td style="width:155px;">G8270</td>
			<td style="width:143px;">Make 20% Stock Solution with MilliQ water; filter sterilize; store at 4 &#176;C; shelf life: 3-6 months. Used to make MCM and MGM.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Sodium pyruvate 100 mM (100x)</td>
			<td style="width:149px;">Hyclone</td>
			<td style="width:155px;">SH30239.01</td>
			<td style="width:143px;">Store at 4 &#176;C. Used to make MCM and MGM.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Penicillin/Streptomycin 10,000 units/ml (100x)</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:155px;">15140-122</td>
			<td style="width:143px;">Store at -20 &#176;C. Used to make MCM, MGM, MM, and DM.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">L-glutamine 200 mM (100x)</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:155px;">25030-081</td>
			<td style="width:143px;">Store at -20 &#176;C. Used to make MCM and MGM.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Fetal Bovine Serum (Defined)</td>
			<td style="width:149px;">Hyclone</td>
			<td style="width:155px;">SH30070.03</td>
			<td style="width:143px;">Filter sterilize; store at -20 &#176;C. Used to make MCM and MGM.</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:233px;">Minimum Essential Medium Earle&#39;s (MEM)</td>
			<td style="width:149px;">Cellgro</td>
			<td style="width:155px;">15-010-CV</td>
			<td style="width:143px;">Without L-glutamine. Contains Earle&#39;s salts. Used to make MCM, MGM, and DM.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Horse Serum</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:155px;">16050</td>
			<td style="width:143px;">Filter sterilize; store at -20 &#176;C. Used to make MCM and MGM.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Hanks&#39; Balanced Salt Solution (HBSS)</td>
			<td style="width:149px;">Cellgro</td>
			<td style="width:155px;">21-021-CV</td>
			<td style="width:143px;">Without calcium and magnesium. Store at 4 &#176;C. Used to make MM.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">HEPES 1 M</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:155px;">15630-031</td>
			<td style="width:143px;">Store at 4 &#176;C. Used to make MM.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">T-75 Flask</td>
			<td style="width:149px;">Corning</td>
			<td style="width:155px;">430641</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">4&#39;,6-Diamidino-2-Phenylindole, dilactate (DAPI)</td>
			<td style="width:149px;">Invitrogen</td>
			<td style="width:155px;">D3571</td>
			<td style="width:143px;">Used to stain cell nucleus.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Rabbit anti-Iba1</td>
			<td style="width:149px;">Wako</td>
			<td style="width:155px;">019-19741</td>
			<td style="width:143px;">Used at 1/750 dilution for ICC staining of Iba1.&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Rabbit anti-NF&#954;B (p65)</td>
			<td style="width:149px;">Abcam</td>
			<td style="width:155px;">7970</td>
			<td style="width:143px;">Used at 1/1250 dilution for ICC staining for p65.</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:233px;">Alexafluor 594 Goat anti-Rabbit IgG (H+L)</td>
			<td style="width:149px;">Invitrogen</td>
			<td style="width:155px;">A11012</td>
			<td style="width:143px;">Used at 1/1000 dilution for visualization of antigen:antibody complex in ICC.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">10 ml Disposable Serological Pipet</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:155px;">13-678-11E</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">50 ml Disposable Centrifuge Tube</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:155px;">05-539-8</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">15 ml Disposible Centrifuge Tube</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:155px;">05-539-12</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Sterile Polystyrene Petri Dish</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:155px;">875713</td>
			<td style="width:143px;">100 mm x 15 mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Scissor: Straight Metzembaum (scissor #1)</td>
			<td style="width:149px;">Roboz Surgical</td>
			<td style="width:155px;">RS-6010</td>
			<td style="width:143px;">1; 5 inch; used for removing head</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Scissor: Vannas 
			(scissor #2)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">15000-08</td>
			<td style="width:143px;">1; non-angled; 2.5mm cutting edge; used to open scalp</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Scissor: Student Vannas (scissor #3)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">91501-09</td>
			<td style="width:143px;">1; curved; used to mince brain tissue</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Forcep: Dumont #7 
			(forcep #1)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">91197-00</td>
			<td style="width:143px;">2; used to secure nose and remove cortices&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Forcep: Dumont #2 
			(forcep #2)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">11223-20</td>
			<td style="width:143px;">1; used to remove scalp</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Forcep: Dumont #3 
			(forcep #3)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">11231-30</td>
			<td style="width:143px;">1; used to remove skull</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Forcep: Dumont #5a 
			(forcep #4)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">11253-21</td>
			<td style="width:143px;">1; used to remove meninges</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Table of specific equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Name of Equipment</td>
			<td style="width:149px;">Name of Company</td>
			<td style="width:155px;">Catalogue Number</td>
			<td style="width:143px;">Comments</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Zoom Stereo Dissection Microscope&#160;</td>
			<td style="width:149px;">Olympus</td>
			<td style="width:155px;">SZ4060</td>
			<td style="width:143px;">Microscope is placed inside Laminar-Horizontal Flow Cabinet</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Laminar-Horizontal Flow Cabinet</td>
			<td style="width:149px;">Nuaire</td>
			<td style="width:155px;">NU-201-330</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Biological Safety Cabinet</td>
			<td style="width:149px;">Labconco</td>
			<td style="width:155px;">3440001</td>
			<td style="width:143px;">Class II</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:233px;">Water-Jacketed CO2 Incubator</td>
			<td style="width:149px;">VWR</td>
			<td>97025-836</td>
			<td style="width:143px;">Set to 37 &#176;C, 5% CO2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Swing-out buckets</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>75006441</td>
			<td style="width:143px;">To be used with Swing-out rotor</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Swing-out Rotor</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>75006445</td>
			<td style="width:143px;">Max Radius: 19.2 (cm)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Sorvall Legend RT+ Centrifuge 
			(clinical centrifuge)</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>75-004-377</td>
			<td style="width:143px;">With swing-out rotor</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">AccuSpin Micro 17 microcentrifuge 
			(tabletop microcentrifuge)</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>S98645</td>
			<td style="width:143px;">With microliter rotor (24 x 1.5/2.0 ml; 
			Cat #: 75003524)&#160;&#160;</td>
		</tr>
	</tbody>
</table>

isolation of cortical microglia with preserved immunophenotype and functionality from murine neonates

El aislamiento de microglia de tejido del SNC es una poderosa herramienta de investigación utilizada para estudiar la biología microglial ex vivo. El presente método detalla un procedimiento para el aislamiento de la microglía de las cortezas murinos neonatales por agitación mecánica con un agitador rotatorio. Este aislamiento microglia método rendimientos microglia corticales altamente puros que exhiben características morfológicas y funcionales indicativos de la microglia en reposo en condiciones normales, no patológicas in vivo. Este procedimiento también preserva el inmunofenotipo de la microglia y la funcionalidad bioquímica como se demuestra por la inducción de cambios morfológicos, la translocación nuclear de la subunidad p65 de NF-kappa B (p65), y la secreción de la citoquina proinflamatoria sello, factor de necrosis tumoral-α (TNF-α ), al lipopolisacárido (LPS) y Pam 3 CSK 4 (Pam) desafíos. Por lo tanto, el presente procedimiento de aislamiento conserva el inmunofenotipo de tanto reposo y Actimicroglia vada, proporcionando un método experimental de la investigación de la biología de la microglía en condiciones ex vivo.

Conservando el inmunofenotipo y la funcionalidad de la microglia ex vivo durante el aislamiento es crítico para ser capaz de utilizar estas células como un modelo de investigación para la biología de la microglia. Con el fin de demostrar la preservación con éxito de immunofunctionality microglia utilizando el presente método, se aislaron microglia corticales de los recién nacidos P3 (CX3CR1-GFP + / - y ratones C57BL / 6) y los cultivos tratados con LPS o Pam. Como...

Watch this Scientific Journal Video about Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates at JoVE.com

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Una clave para el éxito de la investigación de la biología microglial es la preservación de immunofunction microglial ex vivo durante el aislamiento del tejido del SNC. Aislar microglia mediante agitación rotatoria resultados en cultivos de células de alta pureza y inmunofuncional según la evaluación de imágenes fluorescentes, inmunocitoquímica y ELISA después de la activación microglia con el lipopolisacárido estímulos proinflamatorias (LPS) y Pam 3 CSK 4 (Pam).

Aislamiento de cortical Microglia con Inmunofenotipo Conservas y funcionalidad de murinos Neonatos

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

Research

JoVE Journal

Immunology and Infection

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

El aislamiento de la microglía Primaria cultivos mixtos de células gliales del tejido neonatal cerebro de rata

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Investigación

Inmunología e Infección

Isolation, Processing and Analysis of Murine Gingival Cells

El aislamiento, tratamiento y análisis de células gingivales murinos

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia cultivo del Sistema Nervioso Central Neonatal y Adultos

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

El aislamiento, la identificación y purificación de células epiteliales del timo murino

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation of Murine Lymph Node Stromal Cells

Aislamiento de células estromales murinas en nódulos linfáticos

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

Aislamiento y Trasplante de diferentes Aged murinos tímicos injertos.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

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 ...

Isolation and Activation of Murine Lymphocytes

Aislamiento y activación de los linfocitos murinos

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...