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

Bu çalışma NIEHS R01ES014470 (KMZ) tarafından desteklenmiştir.

Önce bu protokolü başlayan, doğum sonrası günlerde yenidoğan farelerin toplamak 1-3 koruma ve sıcaklık için özgün kafes yatak ile iç içe bir steril kap içinde (P1-3). Bu mikrogliyal verimi optimize etmek, bu protokol sayesinde hızlı ve verimli bir şekilde çalışmak önemlidir. Tam reaktif listesi için Tablo 1&#39;e bakınız lütfen. 1.. Aletleri, Kültür, Medya ve Yemekleri hazırlanması <ol><li> 10 ml / pup Mikroglia tam ortam maddesi (MCM hazırla...

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Bu prosedür, neonatal farelerde kortikal mikroglia izolasyonu için etkili bir yöntem sunmaktadır. Bu prosedür, flüoresan görüntüleme, bağışıklık kimyası ve ELISA ile tespit edildiği üzere, 1) koruyucu mikroglia immunfenotipik ve işlevsellik iki kat yararı vardır, ve 2) mikroglia önce diğer glial hücreleri (astrositler ve oligodentrocytes) varlığında, olgun izin glial kültür olgunlaşma döneminde önemli hücre-hücre etkileşimleri kolaylaştırabilir izolasyon. Önemlisi, izole hücreler ko...

Mikroglial hücreleri, CNS parankima gözetim makrofajlar, erişkin memeli beyin, toplam hücre popülasyonunun yaklaşık olarak% 12 ihtiva eder. Mikroglia yolk kesesi miyeloid öncü hücrelerinden türetilen ve yetişkin CNS 1-5 içinde farklı bölgelerde cytoarchitectural hücre yoğunluğu ve morfolojik olarak değişmektedir. Sağlıklı bir yetişkin beyninde, mikrogliya ince, dinamik süreçleri ile, küçük dallanmış veya polar hücrelerdir. Bununla birlikte, in vivo görüntüleme çalışmaları mikroglial işlemleri dinamik &quot;andıran bir şekilde uzanır ve mikro izlemek için geri göstermektedir; çevresel makrofaj morfolojisi aksine, mikroglia hücresel işlem olarak görünebilir sağlıklı beyinlerinde bir hareketsiz, düşük profilli bir fenotip göstermektedir örnekleme ve &quot;6,7 ölçme. Mikroglia geçiş, yüksek ve diferansiyel olarak beyinde çevresel ve patofizyolojik değişikliklere duyarlısırasıyla bir efektör devlet-genellikle kendi dinlenme olarak kabul ve aktif devletler, bir surveillant dan. Aktivasyonuna Bu anahtar, bu patojenle ilişkili moleküler kalıpları yanıt toll benzeri reseptörler (TLR), (PAMPs), yani bakteri ve viral türevli lipoproteinler olarak membrana bağlı görüntü tanıma reseptörleri (PRR) arasında takılma ile aracılık edilebilir , nükleik asitler, karbonhidratlar ve 8-11. PAMPs ek olarak, PRR&#39;ler, aynı zamanda, hücre hasarı 12-16 gibi CNS homeostatisinde sarsımına temsil tehlike / hasar ile bağlantılı moleküler kalıpları (DAMPS) olarak bilinen steril, patojenik olmayan moleküllere karşı, mikroglial aktivasyonunu teşvik ettiği gösterilmiştir. Bir kez yerleştikten sonra PRR mikroglial hücre morfolojisi ve gen ekspresyonunda değişiklikler ile sonuçlanır bir hücre içi sinyalleme kaskadını başlatabilir, özel olarak ise, aktive edilmiş mikroglia bir amoeboid benzeri fenotip adapte hücre çekirdeğine NF-KB p65 alt ünitesi (p65) yerini değiştirmek ve upregüle süredenşeklinde gösterilmektedir ve bu reaktif oksijen türleri (ROS) 16-24 ile birlikte, tümör nekroz faktörü-α (TNF-α), interlökin-1 β (IL-1β) gibi proenflamatuar sitokinlerin salgılanması. CNS&#39;nin doğuştan gelen immün yanıt olarak tamamlayıcı olsa da, bu salgılanan moleküller de bu şekilde, Parkinson ve Alzheimer hastalığı gibi hastalık durumlarının 25-29 nörodejenerasyonu neden olan ve arttıran, nöronal oksidatif stres geliştirmek için bulunmuştur. Bununla birlikte, patolojik durumların mikrogliyal aktivasyonun mekanizması tam olarak anlaşılmış değildir. Mikroglial aktivasyonun vivo özellikleri birçok kültür değinmeyecek olabilir Bu nedenle, mikroglia izolasyonu, bu biyolojik süreçlere güçlü bir araştırma aracıdır. Çeşitli yöntemler CNS doku 30,31 enzimatik sindirimi takip eden bir Percoll gradyanı ile izolasyon dahil olmak üzere, mikroglia, izole edilmesi için kullanılabilir. Bununla birlikte, enzimatik sindirim değiştirebilirAntijen ekspresyonu 32 hücre yüzeyi azaltılması ve burada tarif edilen yönteme göre, hayvan başına daha düşük hücre verimle sonuçlar ile hücrelerin immünofenotipi. Daha önce bir Percoll verim 3-5 x 10 5 hücre 30,33,34 yoluyla tüm CNS izolasyon yöntemleri rapor ederken Özellikle, biz, 7,5 x 10 5 hücre yavru korteksinde başına ortalama mikrogliya verim rapor. Bu prosedür, bu şekilde mikroglial immünfenotip ve işlevselliğini korumak, düşük-yapışma özelliklerine göre mikroglia izole edilmesi ile sindirim enzimlerinin aşılmaktadır. Döner bir karıştırıcı, daha önce bir uzantısına mekanik ajitasyon ile, ve C57BL / 6 faregiller korteks - Bu çalışmada, neonatal heterozigot CX3CR1-GFP (CX3CR1-GFP + /) türetilmiş karışık glial kültürlerinden mikroglial hücreleri izole edilmesini tarif 24,35 yayınlanan yöntem. Biz mikrogliya kolay görsellik için eski fare zorlanma kullanmak gibibir monosit spesifik promoter 36-38 - Bu farenin endojen CX3CR1 mahalinin kontrolü altında GFP ifade eder. Bu yöntem, bir korunmuş immünofenotipi ex vivo bakteriyel lipopolisakkarid (LPS) veya Pam 3 CSK 4 ile meydan morfolojik değişiklikler, p65 nükleer translokasyonu ve TNF-α salgılanması ile gösterildiği gibi, TLR4 ve TLR1 / 2 agonistleri ile son derece saf bir kültür Mikroglial üretir sırasıyla.

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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<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

CNS dokusundan mikrogliya İzolasyonu ex vivo mikrogliyal biyolojisi okumak için kullanılan güçlü bir araştırma aracıdır. Mevcut yöntem, döner bir karıştırıcı, mekanik çalkalama ile neonatal murin kortekslerinden mikroglia izolasyonu için bir prosedürü göstermektedir. Bu yöntem, mikroglia izolasyon verimi canlı içinde normal ve patolojik olmayan koşullarda sakin mikroglia göstergesidir morfolojik ve fonksiyonel özelliklerini gösteren yüksek ölçüde saf bir kortikal mikroglia. Morfolojik değişiklikler, NF-KB (p65) bir p65 alt birimi nükleer translokasyonu ve ayırt edici özelliği pro-enflamatuar sitokin salgılanması, tümör nekroz faktörü-α (TNF-α endüksiyonu ile gösterildiği gibi, bu prosedür, aynı zamanda mikroglial immünfenotip ve biyokimyasal özelliğe korur ), lipopolisakarid&#39;e (LPS) ve Pam 3 CSK 4 (Pam) zorluklar üzerine. Bu yüzden, bu izolasyon prosedürü ve hareketsiz acti hem de immünfenotip korurex vivo koşullarda mikrogliya biyolojiyi araştıran deneysel bir yöntem sağlayan çıkartılır mikrogliya.

Izolasyonu sırasında mikroglia ex vivo immunfenotipine ve işlevselliğini İstinat mikrogliyal biyoloji için bir soruşturma model olarak bu hücreleri kullanmak edebilmek için önemlidir. (- Ve C57BL / 6 CX3CR1-GFP + /) ve LPS veya Pam ya ile tedavi edilen kültürler, bu yöntem kullanılarak mikroglia immunofunctionality başarılı bir şekilde korunması göstermek amacıyla, P3 yenidoğanlardan kortikal mikroglia izole edilmiştir. Şekil 1A&...

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

Mikrogliyal biyoloji başarılı bir soruşturma için bir anahtar CNS dokudan izolasyonu sırasında ex vivo mikroglialardan immunofunction korunmasıdır. Flüoresan görüntüleme, bağışıklık kimyası ve proenflamatuar stimuli lipopolisakarit (LPS) ile Pam 3 CSK 4 (PAM) ile ELISA aşağıda mikroglia aktivasyonu ile değerlendirilen gibi son derece saf ve immunofunctional hücre kültürlerinde döner çalkalama sonuçları ile mikroglia izole edilmesi.

Kemirgen Yenidoğan itibaren Korunan immunfenotipine ve İşlevselliği ile Kortikal mikrogliya İzolasyonu

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

Yenidoğan Rat Beyin Dokusu Karışık Glial Hücre Kültürleri Primer mikroglia İzolasyonu

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

Murin Dişeti Hücre İzolasyonu, İşleme ve Analiz

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

Yenidoğan ve Erişkin Merkezi sinir sisteminin Kültüre Mikroglia

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

İzolasyon, tanımlama ve Murin Thymic Epitel Hücreleri saflaştırılması

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

Kemirgen Lenf Düğümü Stromal Hücrelerinin İzolasyonu

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.

İzolasyon ve Farklı Yaşlı Fare Timik Greft Nakli.

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

Maternal Fetal Arayüz at Fare Dokular lökosit İzolasyonu

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

İzolasyon ve murin lenfositlerin aktivasyonu

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