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

Detta arbete stöddes av NIEHS R01ES014470 (KMZ).

Före start detta protokoll, samla neonatala möss vid postnatal dag 1-3 (P1-3) i en steril behållare kapslad med original bur strö för skydd och värme. Det är viktigt att arbeta snabbt och effektivt genom detta protokoll för att optimera microglial avkastning. Se tabell 1 för fullständig reagens lista. 1. Beredning av instrument, Kultur Media och rätter <ol><li> Bered 10 ml / pup Mikroglia Complete Media (MCM; 1x Minimal Essential Medium Earles [MEM] kompletteras...

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Det nuvarande förfarandet erbjuder en effektiv metod för isolering av kortikala mikroglia från neonatala möss. Detta förfarande har en dubbel nytta av 1) bevara mikroglia immunofenotyp och funktionalitet, som bestäms av fluorescerande imaging, immuncytokemiundersökning och ELISA, samt, 2) låta mikroglia att mogna i närvaro av andra gliaceller (astrocyter och oligodentrocytes) före isolering, som kan underlätta viktiga cell-cell-interaktioner under glial kultur mognadsperiod. Viktigt är isolerade celler visar...

Mikrogliaceller, övervaknings makrofagerna i CNS parenkym, utgör ungefär 12% av den totala cellpopulationen av den vuxna däggdjurshjärnan. Mikroglia härrör från gulesäcken myeloid precursorceller och varierar i celltäthet och morfologi på olika cytoarchitectural regioner inom den vuxna CNS 1-5. I en frisk vuxen hjärna, mikroglia är små, förgrenade eller polära celler med fina, dynamiska processer. I motsats till perifera makrofager morfologi, mikroglia visar en vilande, låg profil fenotyp i friska hjärnor som kan visas som cellulär inaktivitet, men in vivo imaging studier visar att microglial processer dynamiskt sträcka ut och dra för att övervaka deras mikromiljö på ett sätt som påminner om &quot; provtagning och mätning &quot;6,7. Mikroglia är starkt och differentiellt lyhörda för miljö-och patofysiologiska förändringar i hjärnan, bytafrån en surveillant till en effektor stats allmänt betraktas som deras vilande och aktiverade stater, respektive. Denna switch i aktivering kan förmedlas genom ingrepp av membranbundna mönsterigenkänningsreceptorer (PRRS), såsom avgiftsliknande receptorer (TLRs), som svarar mot patogen-associerade molekylära mönster (PAMPs), nämligen bakterie-och virus-härledda lipoproteiner , nukleinsyror och kolhydrater 8-11. Förutom PAMPs har PRRs även visats inducera microglial aktivering mot sterila, icke-patogena molekyler som kallas fara / skada-associerade molekylära mönster (dämpas), som utgör en störning i CNS homeostas, såsom cellskador 12-16. En gång i ingrepp, PRRs initiera en intracellulär signalkaskad som resulterar i förändringar i microglial cellmorfologi och genexpression; specifikt aktiverade mikroglia anpassa en amöboid liknande fenotyp, translokera p65-NF-KB-subenhet (p65) till cellkärnan, och uppreglera produInsatser och utsöndring av proinflammatoriska cytokiner, såsom tumörnekrosfaktor-α (TNF-α), interleukin-1 β (IL-1β), tillsammans med reaktiva syreradikaler (ROS) 16 till 24. Även integrerad i det medfödda immunsvaret i CNS har dessa utsöndrade molekyler även befunnits öka neuronal oxidativ stress och därmed inducera och förvärrar neurodegeneration i sjukdomstillstånd som Parkinsons och Alzheimers sjukdom 25-29. Emellertid är mekanismen av microglial aktivering i patologiska tillstånd inte helt klarlagd. Därför är isolering av mikroglia ett kraftfullt undersökande verktyg i dessa biologiska processer, så många in vivo funktioner i microglial aktivering kan rekapituleras i kultur. Flera metoder finns tillgängliga för isolering av mikroglia, inklusive isolering via en Percoll gradient efter enzymatisk nedbrytning av CNS-vävnad 30,31. Emellertid kan enzymatisk spjälkning förändraden immunofenotyp av cellerna genom att minska cellyteantigen uttryck 32 och resulterar i lägre cellutbyte per djur än den metod som beskrivs häri. Specifikt rapporterar vi en genomsnittlig mikroglia avkastning per valp cortex av 7,5 x 10 5 celler, medan tidigare rapporterade isoleringsmetoder från hela CNS via en Percoll gradient avkastning 3-5 x 10 5 celler 30,33,34. Det nuvarande förfarandet kringgår användning av matsmältningsenzymer genom att isolera mikroglia baserat på deras låg vidhäftningsförmåga och därmed bevara den microglial immunofenotyp och funktionalitet. I den aktuella studien beskriver vi isoleringen av mikrogliaceller från blandade gliaceller kulturer härrör från neonatal heterozygota CX3CR1-GFP (CX3CR1-GFP + / -) och C57BL / 6 mus cortex genom mekanisk omrörning på en roterande shaker, en förlängning av tidigare publicerad metod 24,35. Vi utnyttjar det tidigare musstam för enkel visualisering av mikroglia, somDessa möss uttrycker GFP under kontroll av den endogena CX3CR1 locus - en monocyt-specifik promotor från 36 till 38. Denna metod ger högrena microglial kulturer med en bevarad immunofenotyp ex vivo såsom visas genom morfologiska förändringar, nukleär translokation av p65 och utsöndring av TNF-α när de utsattes för bakteriell lipopolysackarid (LPS) eller Pam 3 CSK 4, TLR4 och TLR1 / 2 agonister , respektive.

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		<tr height="160">
			<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

Isolering av mikroglia från CNS-vävnad är ett kraftfullt utredningsverktyg som används för att studera microglial biologi ex vivo. Den nuvarande metoden information ett förfarande för isolering av mikroglia från neonatal mus cortex genom mekanisk omrörning med en roterande skak. Denna mikroglia isolering metod ger mycket rena kortikala mikroglia som uppvisar morfologiska och funktionella egenskaper tyder på vilande mikroglia i normala, nonpathological förhållanden in vivo. Detta förfarande bevarar också den microglial immunofenotyp och biokemisk funktion som visas av inducering av morfologiska förändringar, nukleär translokation av p65-subenheten hos NF-kB (p65) och utsöndring av ett kännetecken proinflammatorisk cytokin, tumörnekrosfaktor-α (TNF-α ), på lipopolysackarid (LPS) och Pam 3 CSK 4 (Pam) utmaningar. Därför bevarar immunofenotyp av både vilande och akti föreliggande isoleringsprocedurverad mikroglia, som ger en experimentell metod för att undersöka mikroglia biologi i ex vivo-förhållanden.

Att behålla immunofenotyp och funktionaliteten av mikroglia ex vivo under isoleringen är avgörande för att kunna utnyttja dessa celler som en utredningsmodell för microglial biologi. För att demonstrera den framgångsrika bevarandet av mikroglia immunofunctionality användning av föreliggande förfarande isolerade vi kortikala mikroglia från P3 nyfödda (CX3CR1-GFP + / - och C57BL / 6) och behandlades kulturerna med antingen LPS eller Pam. Såsom illustreras i <st...

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

En nyckel till framgångsrik utredning av microglial biologi är bevarandet av microglial immunofunction ex vivo under isolering från CNS-vävnad. Isolera mikroglia via roterande skakar resultat i mycket rena och immunofunctional cellkulturer som bedöms av fluorescerande imaging, immuncytokemiundersökning och ELISA efter mikroglia aktivering med proinflammatoriska stimuli lipopolysackarid (LPS) och Pam 3 CSK 4 (Pam).

Isolering av kortikal Mikroglia med Konserverade immunofenotyp och funktionalitet från Murina Nyfödda

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

Primär Mikroglia Isolering från Blandade gliaceller Kulturer av nyfödda råttor Brain Tissue

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

Isolering, bearbetning och analys av murina 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

Odling Mikroglia från neonatal och vuxna centrala nervsystemet

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

Isolering, identifiering och rening av murina Thymic epitelceller

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

Isolering av murint Lymph Node stromaceller

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.

Isolering och transplantation av olika Aged Murina tymisk ympkvistar.

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

Isolering av leukocyter från Murina vävnader på 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

Isolering och aktivering av murina lymfocyter

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