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

Dit werk werd ondersteund door NIEHS R01ES014470 (KMZ).

Voorafgaand aan de start van dit protocol, verzamelen neonatale muizen op postnatale dagen 1-3 (P1-3) in een steriele container genest met originele kooi beddengoed voor bescherming en warmte. Het is belangrijk om snel en efficiënt te werken door middel van dit protocol om microglia opbrengst te optimaliseren. Zie tabel 1 voor volledige reagenslijst. 1. Voorbereiding van de instrumenten, Cultuur Media en Gerechten <ol><li> Bereid 10 ml / pup Microglia compleet medium (MCM...

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De huidige procedure biedt een effectieve methode voor het isoleren van de corticale microglia van pasgeboren muizen. Deze procedure heeft een dubbel voordeel 1) behoud microglia immunophenotype en functionaliteit, zoals bepaald door fluorescentie beeldvorming, immunocytochemie en ELISA, en 2) zodat microglia rijpen in aanwezigheid van andere gliacellen (astrocyten en oligodentrocytes) vóór isolatie, die belangrijke cel-cel interacties tijdens de gliale cultuur rijpingsduur kan vergemakkelijken. Belangrijk, geïsoleer...

Microgliacellen, het toezicht macrofagen van het CZS parenchym, omvatten ongeveer 12% van de totale celpopulatie van de volwassen hersenen van zoogdieren. Microglia zijn afgeleid uit dooierzak myeloïde voorlopercellen en variëren in celdichtheid en morfologie in verschillende cytoarchitectural gebieden binnen de volwassen CZS 1-5. In een gezonde volwassen brein, microglia zijn kleine, vertakte of polaire cellen met fijne, dynamische processen. In tegenstelling tot perifere macrofagen morfologie, microglia tonen een rustige, low-profile fenotype in gezonde hersenen die kunnen worden weergegeven als cellulaire inactiviteit, maar in vivo imaging studies tonen aan dat microglia processen dynamisch en uitschuiven om hun micro-omgeving te controleren op een manier die doet denken aan &quot; bemonstering en landmeetkundige &quot;6,7. Microglia en zijn zeer verschillend reageren op milieu en pathofysiologische veranderingen in de hersenen, switchingeen toezichthouder van een effector-staat algemeen beschouwd als de rustende en geactiveerde toestanden, respectievelijk. Deze schakelaar activering kan worden gemedieerd door aangrijping van membraangebonden patroonherkenning receptoren (PRRS), zoals toll-like receptoren (TLR&#39;s), die beantwoorden aan pathogeen-geassocieerde moleculaire patronen (PAMPs), namelijk bacteriële-en virale afgeleide lipoproteïnen , nucleïnezuren en koolhydraten 8-11. Naast PAMPs zijn PRRS ook aangetoond dat microglia activatie induceren tegen steriel, niet-pathogene moleculen, bekend als gevaar / beschadiging geassocieerde moleculaire patronen (gedempt), die een verstoring in CZS homeostase vertegenwoordigen, zoals cellulaire schade 12-16. Eenmaal geactiveerd, PRRS initiëren een intracellulaire signalerende cascade die leidt tot veranderingen in microgliale cel morfologie en genexpressie, specifiek geactiveerde microglia aan een amoeboid fenotype, verplaatsen de P65 subeenheid NF-kB (p65) naar de celkern, en de upregulate productie en secretie van pro-inflammatoire cytokinen zoals tumornecrosefactor-α (TNF-α), interleukine-1 β (IL-1β), samen met reactieve zuurstof species (ROS) 16-24. Hoewel integraal in de aangeboren immuunrespons van het CZS, zijn deze afgescheiden moleculen ook gevonden neuronale oxidatieve stress te vergroten, waardoor het induceren en verergeren neurodegeneratie in zieke toestanden zoals Parkinson en Alzheimer 25-29. Echter, de mechanismen van microgliale activering in pathologische toestanden niet volledig begrepen. Daarom isolatie van microglia is een krachtig onderzoeksinstrument in deze biologische processen, zoals velen in vivo functies van microglia activatie kan worden samengevat in de cultuur. Verschillende methoden beschikbaar voor het isoleren van microglia, waaronder isolatie via een Percoll gradiënt na enzymatische afbraak van CZS weefsel 30,31. Echter, enzymatische digestie veranderenhet immunofenotype van de cellen door het verminderen celoppervlak antigeen-expressie 32 en resulteert in lagere cel opbrengst per dier dan de hierin beschreven werkwijze. Specifiek, melden wij een gemiddelde microglia opbrengst per pup cortex van 7,5 x 10 5 cellen, terwijl eerder gemeld isolatie methoden uit geheel CNS via een Percoll gradiënt opbrengst 3-5 x 10 5 cellen 30,33,34. De huidige procedure omzeilt het gebruik van de spijsvertering enzymen door het isoleren van microglia op basis van hun lage-aanhankelijkheid eigenschappen, waardoor de microglia immunofenotype en functionaliteit behoud. In deze studie beschrijven we de isolatie van microgliale cellen uit gemengde gliale kweken afgeleid van neonatale heterozygote CX3CR1-GFP (CX3CR1-GFP + / -) en C57BL / 6 muizen cortex door mechanische agitatie op een roterende schudder, een uitbreiding van eerder gepubliceerd methode 24,35. We maken gebruik van de voormalige muizenstam voor eenvoudige visualisatie van microglia, zoalsDeze muizen brengen GFP onder de controle van de endogene locus CX3CR1 - een monocyt-specifieke promotor 36-38. Deze werkwijze levert zeer zuiver microgliale kweken met een behouden immunofe ex vivo zoals blijkt uit morfologische veranderingen, nucleaire translocatie van p65 en uitscheiding van TNF-α bij provocatie met bacteriële lipopolysaccharide (LPS) of Pam 3 CSK 4, TLR4 en TLR1 / 2 agonisten respectievelijk.

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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

Isolatie van microglia van CZS-weefsel is een krachtig onderzoeksinstrument gebruikt om microglia biologie studeren ex vivo. De huidige methode Gegevens van een procedure voor de isolatie van microglia van neonatale muizen cortex door mechanisch roeren met een roterende schudder. Dit microglia isolatie methode levert zeer zuiver corticale microglia dat morfologische en functionele kenmerken wijzen op latente microglia in normale, nonpathological condities in vivo vertonen. Deze procedure bewaart ook de microgliale immunofenotype en biochemische functies zoals blijkt uit de inductie van morfologische veranderingen, nucleaire translocatie van de p65 subeenheid van NF-kB (p65), en secretie van de pro-inflammatoire cytokine kenmerk, tumornecrosefactor-α (TNF-α ), na lipopolysaccharide (LPS) en Pam 3 CSK 4 (Pam) uitdagingen. Daarom is de huidige isolatie procedure behoudt de immunofenotype van zowel latente en actikeld microglia, die een experimentele methode van onderzoek microglia biologie in ex vivo omstandigheden.

Behoud van immunofenotype en functionaliteit van microglia ex vivo tijdens isolatie kritisch kunnen deze cellen te gebruiken als onderzoeks model voor microgliale biologie. Om de succesvolle behoud van microglia immunofunctionality volgens de onderhavige werkwijze demonstreren, isoleerden wij corticale microglia van P3 pasgeborenen (CX3CR1-GFP + / - en C57BL / 6) en behandelde kweken met ofwel LPS of Pam. Zoals getoond in figuur 1A, isolatie van microglia...

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

Een sleutel tot succesvolle onderzoek van microglia biologie is het behoud van microglia immunofunction ex vivo tijdens isolatie van CZS-weefsel. Isoleren microglia via roterende schudden resulteert in zeer zuivere en immunofunctional celculturen zoals beoordeeld door fluorescerende beeldvorming, immunocytochemie, en ELISA na microglia-activatie met de pro-inflammatoire stimuli lipopolysaccharide (LPS) en Pam 3 CSK 4 (Pam).

Isolatie van corticale Microglia met Conserven immunofenotype en functionaliteit van Muriene Pasgeborenen

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

Primaire Microglia Isolatie van Mixed gliale celculturen van neonatale rat hersenweefsel

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

Isolation, Processing and Analysis of Murine Gingival Cells

Isolatie, verwerking en analyse van Muriene Gingival Cells

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

Kweken Microglia van de neonatale en volwassen centrale zenuwstelsel

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

Isolatie, identificatie en zuivering van Muriene thymus epitheelcellen

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

Isolatie van muizen lymfkliertest stromale cellen

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.

Isolatie en Transplantatie van verschillende Aged Muriene Thymic Enten.

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

Isolatie van leukocyten uit de Murine Tissues de Maternal-Fetal Interface

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

Isolatie en Activering van Muizen lymfocyten

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

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Analyse van lymfocyt extravasatie gebruik van een<em&gt; In Vitro</em&gt; Model van het menselijk bloed-hersenbarrière

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Isolatie van extracellulaire blaasjes aan de lymfkliertest Bronchoalveolar Lavage vloeistof met behulp van een techniek van ultrafiltratie centrifugeren