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 arbeidet ble støttet av NIEHS R01ES014470 (KMZ).

Før du starter denne protokollen, samle neonatal mus ved døgnene 1-3 (P1-3) i en steril beholder nestet med originale bur sengetøy for beskyttelse og varme. Det er viktig å jobbe raskt og effektivt gjennom denne protokollen for å optimalisere microglial yield. Vennligst se tabell 1 for fullstendig reagenslisten. En. Utarbeidelse av instrumenter, Culture Media og retter <ol><li> Forbered 10 ml / pup Microglia Komplett Media (MCM, 1x Minimal Essential Medium Earles [MEM...

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Den foreliggende fremgangsmåte gir en effektiv metode for isolering av kortikale microglia fra nyfødte mus. Denne prosedyren har en to-fold fordel for en) bevare mikroglia immunophenotype og funksjonalitet, som bestemmes av fluorescerende bildebehandling, immunocytochemistry, og ELISA, og, 2) slik at mikroglia å modne i nærvær av andre gliacellene (astrocytes og oligodentrocytes) før isolering, noe som kan forenkle viktige celle-celle-vekselvirkninger under glial kultur modningsperiode. Viktigere, isolerte celler ...

Mikrogliale celler, makrofager overvåking av CNS-parenchyma, utgjør omtrent 12% av den totale cellepopulasjon av den voksne hjernen hos pattedyr. Microglia er utledet fra plommesekken myeloide forløper celler og varierer i celletetthet og morfologi i ulike cytoarchitectural regioner innenfor voksen CNS 1-5. I en frisk voksen hjerne, microglia er små, ramified eller polare celler med fine, dynamiske prosesser. I motsetning til perifer makrofag morfologi, mikroglia demonstrere en sovende, lavprofil fenotype hos friske hjerner som kan vises som cellular inaktivitet, men in vivo imaging studier viser at microglial prosesser dynamisk ut og inn for å overvåke deres mikromiljøet på en måte som minner om &quot; prøvetaking og oppmåling &quot;6,7. Microglia er høyt og forskjellig lydhør for miljø-og patofysiologiske forandringer i hjernen, byttefra en surveillant til en effektor stats ofte regnet som deres hvile og aktiveres stater, henholdsvis. Denne bryter i aktiveringen kan være mediert ved inngrep av membran-bundet mønstergjenkjenning reseptorer (PRRs), for eksempel toll-lignende reseptorer (TLR), som reagerer på patogen-assosiert molekyl mønstre (PAMPs), nemlig bakterie-og virus-avledede lipoproteiner , nukleinsyrer, karbohydrater og 8-11. I tillegg til PAMPs, har PRRs også blitt vist å indusere microglial aktivering mot sterilt, ikke-patogeniske molekyler som kalles fare / skadeassosiert molekyl mønstre (demper), som representerer en forstyrrelse i sentralnervesystemet homeostase, slik som cellulær skade 12-16. Når engasjert, PRRs initiere en intracellulær signalering kaskade som resulterer i endringer i microglial cellemorfologi og genuttrykk, spesielt, aktiveres mikroglia tilpasse en amoeboid lignende fenotype, translocate den p65 NF-κB subenhet (p65) til cellekjernen, og oppregulere proDette skjer og sekresjon av proinflammatoriske cytokiner, slik som tumornekrosefaktor-α (TNF-α), interleukin-1-β (IL-1β), sammen med reaktive oksygenarter (ROS) 16-24. Selv integrert i den medfødte immunrespons i CNS, har disse utskilte molekylene også blitt funnet å øke neuronal oksidativt stress, og dermed indusere og forverrer neurodegenerering i syke tilstander som Parkinsons og Alzheimers sykdom 25-29. Imidlertid er mekanismene for microglial aktivering i patologiske tilstander er ikke helt forstått. Derfor, isolering av microglia er en kraftig undersøkende verktøy inn i disse biologiske prosesser, som mange i vivo funksjoner i microglial aktivering kan recapitulated i kultur. Flere metoder er tilgjengelige for å isolere mikroglia, inkludert isolasjon via en Percoll gradient etter enzymatisk fordøyelse av CNS vev 30,31. Imidlertid kan enzymatisk fordøyelse alterimmunfenotypen av cellene ved å redusere celle-overflate-antigen ekspresjon 32, og resulterer i lavere celleproduksjonen per dyr enn den metode som er beskrevet heri. Nærmere bestemt, rapporterer vi en gjennomsnittlig mikroglia avkastning per valp cortex av 7,5 x 10 5 celler, mens tidligere rapportert isolasjon metoder fra hele CNS via en Percoll gradient avkastning 3-5 x 10 5 celler 30,33,34. Den nåværende prosedyren omgår bruk av fordøyelsesenzymer ved å isolere mikroglia basert på deres lav-overholdelse egenskaper, og dermed bevare microglial immunfenotypen og funksjonalitet. I den foreliggende studien, beskriver vi isolering av microglial celler fra blandede glial kulturer som stammer fra neonatal heterozygote CX3CR1-GFP (CX3CR1-GFP + / -), og C57BL / 6 murine ekser via mekanisk agitasjon på en roterende shaker, en forlengelse av tidligere publisert metode 24,35. Vi utnytter den tidligere mus belastning for enkel visualisering av microglia, somDisse mus uttrykker GFP under kontroll av den endogene Cx3Cr1 locus - en monocytt-spesifikk promoter 36-38. Denne metoden gir svært rene microglial kulturer med en bevart immunophenotype ex vivo som demonstrert av morfologiske endringer, kjernefysisk translokasjon av p65, og sekresjon av TNF-α når utfordret med bakteriell lipopolysakkarid (LPS) eller Pam 3 CSK 4, TLR4 og TLR1 / 2 agonister , henholdsvis.

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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 av mikroglia fra CNS vev er en kraftig undersøkende verktøy som brukes til å studere microglial biologi ex vivo. Den foreliggende fremgangsmåte detaljer av en fremgangsmåte for isolering av mikroglia fra neonatale murine cortices ved mekanisk omrøring med en rotasjonsrister. Dette microglia isolasjon metoden gir svært rene kortikale microglia som viser morfologiske og funksjonelle egenskaper som tyder på hvil mikroglia i normale, nonpathological forhold i vivo. Denne fremgangsmåten bevarer også den microglial immunfenotype og biokjemiske funksjoner som vist ved induksjon av morfologiske endringer, nukleær translokasjon av p65 subenhet av NF-κB (p65) og sekresjon av kjennemerket proinflammatorisk cytokin, tumor nekrose faktor-α (TNF-α ), på lipopolysakkarid (LPS) og Pam 3 CSK 4 (Pam) utfordringer. Derfor bevarer dagens isolasjon prosedyren immunophenotype av både stillestående og aktivigende microglia, som gir en eksperimentell metode for å undersøke microglia biologi i ex vivo forhold.

Beholde immunophenotype og funksjonalitet av mikroglia ex vivo under isolasjon er avgjørende for å kunne utnytte disse cellene som en utredningsmodell for microglial biologi. For å vise den vellykkede bevaring av microglia immunofunctionality ved hjelp av den foreliggende fremgangsmåte, har vi isolert kortikale mikroglia fra P3 nyfødte (CX3CR1-GFP + / - og C57BL / 6) og behandlede kulturer med enten LPS eller Pam. Som vist i figur 1A, isolering av mi...

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 nøkkel til vellykket etterforskning av microglial biologi er bevaring av microglial immunofunction ex vivo under isolasjon fra CNS vev. Isolere mikroglia via roterende risting resultater i svært rene og immunofunctional cellekulturer som vurderes av fluorescerende bildebehandling, immunocytochemistry, og ELISA følgende microglia aktivering med proinflammatoriske stimuli lipopolysakkarid (LPS) og Pam 3 CSK 4 (Pam).

Isolering av Cortical Microglia med Bevarte Immunfenotype og funksjonalitet 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

Primær microglia Isolasjon fra Blandede glial cellekulturer fra Neonatal Rat hjernevev

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, bearbeiding og analyse av 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

Dyrking Microglia fra Neonatal og voksne sentralnervesystemet

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

Isolasjon, Identifikasjon, og rensing av Muse thymisk 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 stromale celler

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.

Isolasjon og Transplantasjon av forskjellige alderen Murine tymisk grafts.

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 leukocyttene fra Murine Vev 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 og aktivering av lymfocytter Murine

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