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

Dette arbejde blev støttet af NIEHS R01ES014470 (KMZ).

Før du starter denne protokol, indsamle neonatale mus på postnatal dag 1-3 (P1-3) i en steril beholder indlejret med originale bur strøelse til beskyttelse og varme. Det er vigtigt at arbejde hurtigt og effektivt gennem denne protokol at optimere microglial udbytte. Se Tabel 1 for komplet liste over reagenser. 1.. Udarbejdelse af instrumenter, Kultur Medier og skåle <ol><li> Forbered 10 ml / pup Mikroglia Complete Media (MCM, 1x Minimal Essential Medium Earles [MEM] su...

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Den nuværende procedure giver en effektiv metode til isolering af kortikale microglia fra neonatale mus. Denne fremgangsmåde har en dobbelt fordel af 1) bevarelse microglia immunofænotype og funktionalitet, som bestemt af fluorescerende billeddannelse, immuncytokemi og ELISA, og 2) at tillade mikroglia at modne i overværelse af andre gliaceller (astrocytter og oligodentrocytes) forud for isolation, hvilket kan fremme vigtige celle-celle interaktioner under glial kultur modningsperiode. Vigtigere er det, udstillet is...

Mikrogliaceller, overvågnings makrofager i CNS parenkym omfatte omkring 12% af den samlede cellepopulation pattedyrhjernen voksen. Mikroglia er afledt fra blommesækken myeloide precursorceller og varierer i celledensitet og morfologi i forskellige cytoarchitectural regioner i den voksne CNS 1-5. I en sund voksen hjerne, microglia er små, forgrenede eller polære celler med fine, dynamiske processer. I modsætning til perifer makrofag morfologi, mikroglia demonstrere en hvilende, lavprofil fænotype hos raske hjerner, der kan fremstå som cellulær inaktivitet, men in vivo billeddiagnostiske undersøgelser viser, at microglial processer dynamisk udvide og trække for at overvåge deres mikromiljø på en måde, der minder om &quot; prøveudtagning og landmåling &quot;6,7. Microglia er meget og forskelligt lydhøre over for miljø-og patofysiologiske forandringer i hjernen, skiftefra en surveillant til en effektor statslige almindeligvis betragtes som deres hvilende og aktiverede tilstande, hhv. Denne kontakt vil i aktivering kan medieres ved indgreb af membranbundne mønstergenkendelse receptorer (PRRS), såsom toll-lignende receptorer (TLRs), som svarer til patogen-associeret molekylære mønstre (PAMPs), nemlig bakterielle-og viral-afledte lipoproteiner , nukleinsyrer og kulhydrater 8-11. Udover PAMPs har PRRs også vist sig at inducere microglial aktivering mod sterile, nonpathogenic molekyler kaldet fare / skade-associeret molekylære mønstre (dæmper), som repræsenterer en perturbation i CNS homeostase, såsom cellulære skader 12-16. Når det er aktiveret, PRRs indlede en intracellulær signalering kaskade, der resulterer i ændringer i mikroglial celle morfologi og genekspression, specifikt aktiverede mikroglia tilpasse et amoeboid-lignende fænotype translokere den p65 NF-KB-underenhed (p65) til cellekernen og opregulere produIndsatsen og sekretion af proinflammatoriske cytokiner, såsom tumornekrosefaktor-α (TNF-α), interleukin-1 β (IL-1β), sammen med reaktive oxygenarter (ROS) 16-24. Selv integreret i immunresponset i CNS medfødte har disse secernerede molekyler også fundet at øge neuronal oxidativ stress, for derved at inducere og forværrer neurodegeneration i sygdomstilstande såsom Parkinsons og Alzheimers sygdom 25-29. Dog er mekanismerne for microglial aktivering i patologiske tilstande ikke helt forstået. Derfor isolering af mikroglia er et kraftfuldt undersøgende værktøj ind i disse biologiske processer, som mange in vivo funktioner i microglial aktivering kan sammenfattet i kultur. Der findes flere metoder til isolering af mikroglia, herunder isolering via en Percoll-gradient efter enzymatisk fordøjelse af CNS-væv 30,31. Dog kan enzymatisk fordøjelse ændreden immunofænotype af cellerne ved at reducere celleoverfladeantigen ekspression 32, og resulterer i lavere celleudbytte per dyr end den heri beskrevne fremgangsmåde. Konkret rapporterer vi en gennemsnitlig microglia udbytte pr hvalp cortex på 7,5 x 10 5 celler, mens tidligere rapporteret isolation metoder fra hele CNS via en Percoll gradient udbytte 3-5 x 10 5 celler 30,33,34. Den nuværende procedure omgår brug af fordøjelsesenzymer ved at isolere microglia baseret på deres lave adhærensegenskaber og dermed bevare den microglial immunofænotype og funktionalitet. I den foreliggende undersøgelse beskriver vi isolering af mikrogliaceller fra blandede gliaceller kulturer stammer fra neonatale heterozygote CX3CR1-GFP (CX3CR1-GFP + / -) og C57BL / 6 murine cortex via mekanisk agitation på en rotationsryster, en udvidelse af tidligere publiceret metode 24,35. Vi udnytter den tidligere musestamme til nem visualisering af mikroglia somdisse mus udtrykker GFP under kontrol af den endogene CX3CR1 locus - en monocyt promotor 36-38. Denne metode frembringer meget rene microgliale kulturer med et konserveret immunofænotype ex vivo som påvist ved morfologiske ændringer, nuklear translokation af p65, og sekretion af TNF-α når udfordret med bakterielt lipopolysaccharid (LPS) eller Pam 3 CSK 4, TLR4 og TLR1 / 2 agonister hhv.

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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

Isolering af mikroglia fra CNS væv er et kraftfuldt efterforskningsmæssige værktøj, der anvendes til at studere microglial biologi ex vivo. Den nuværende metode beskriver en procedure til isolering af mikroglia fra neonatale murine cortex ved mekanisk omrøring med en orbitalryster. Denne microglia isolation metode giver meget rene kortikale mikrogliaceller der udviser morfologiske og funktionelle egenskaber indikerer hvilende microglia i normale nonpathological forhold in vivo. Denne procedure bevarer også microglial immunofænotype og biokemisk funktionalitet som påvist ved induktion af morfologiske ændringer, nuklear translokation af p65-underenheden af ​​NF-KB (p65) og sekretion af kendetegnende proinflammatorisk cytokin, tumornekrosefaktor-α (TNF-α ) efter lipopolysaccharid (LPS) og Pam 3 CSK 4 (Pam) udfordringer. Den foreliggende isoleringsprocedure bevarer immunofænotype både hvilende og aktiverede microglia, hvilket giver en eksperimentel metode til at undersøge mikroglia biologi i ex vivo.

Bevarelse af den immunofænotype og funktionaliteten af mikroglia ex vivo under isolation er afgørende at være i stand til at udnytte disse celler som en undersøgelsesgruppe model for microglial biologi. For at demonstrere en vellykket bevarelse af mikroglia immunofunctionality ved hjælp af den nuværende metode, vi isolerede kortikale microglia fra P3 nyfødte (CX3CR1-GFP + / - og C57BL / 6) og behandlede kulturer med enten LPS eller Pam. Som illustreret i fi...

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 af nøglerne til en vellykket efterforskning af microglial biologi er bevarelsen af microglial immunofunction ex vivo under isolation fra CNS-væv. Isolerende microglia via roterende rystning resulterer i stærkt rene og immunofunktionel cellekulturer som vurderet af fluorescerende billeddannelse, immuncytokemi, og ELISA efter mikroglia aktivering med det proinflammatoriske stimuli (LPS) og Pam 3 CSK 4 (Pam).

Isolering af Cortical Mikroglia med bevarede immunofænotype og funktionalitet fra murine Nyfødte

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

Primære Mikroglia Isolering af blandede gliacelle Kulturer af neonatal rottehjerne 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

Isolation, behandling og analyse af murine 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

Dyrkning Mikroglia fra Neonatal og Adult Central Nervous System

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, identifikation og oprensning af murine thymusepitelmonolag Celler

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 af murine lymfeknuder stromacellers

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 og transplantation af forskellige Aged murine Thymic udtagne transplantater.

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 af leukocytter fra det murine væv på maternel-føtal grænseflade

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 og aktivering af murine lymfocytter

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