Name: isolation and culture of primary neurons and glia from adult rat urinary bladder
Uploaded: 2020-05-23T14:45:00.000+00:00
Duration: 6 min 18 s
Description: Watch this Scientific Journal Video about Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder at JoVE.com

Bu çalışma Çin Ulusal Doğa Bilimleri Vakfı (Grant no. 81673676) ve Dongguan Bilim ve Teknoloji Bürosu (Grant no. 2019622101002) tarafından desteklendi. Yazarlar teknik danışmanlık için Dr. Maryrose Sullivan'a (Harvard Tıp Fakültesi Cerrahi Yardımcı Doçenti) teşekkür eder.

Tüm deneysel protokoller ve hayvan prosedürleri Ulusal Araştırma Konseyi'nin etik ilke yönergelerine uygun olarak yapıldı.
1. Malzemelerin hazırlanması
<ol><li>Deneyi yapmadan önce bir otoklav kullanarak tüm aletleri ve ddH2O'yu sterilize edin. Aletler cerrahi makas, oftalmik makas, hardallar, kaşık çekirdeği bölücü, cam tabaklar (60-100 mm çapında) ve cam kırıcılar içerir ancak bunlarla sınırlı değildir.</li>
	<li>Krebs çözeltisini aşağıdaki gibi hazırlayın (Tablo 1): Kullanmadan önce tüm kimyasalları ddH2O ile birlikte çözün. CaCI2'yi ayrı ayrı çözün ve tortuyu önlemek için karıştırırken karışık çözeltiye yavaşça ekleyin.</li>
	<li>%10 fetal sığır serumu (FBS) ve %1 antibiyotik/antimycotic (100x) ile F12 ortamlarından oluşan Durulama medyasını hazırlayın. 44,5 mL F12 ortama 5 mL FBS ve 0,5 mL antibiyotik/antimikotik ekleyin.</li>
	<li>%2 B-27, %1 FBS, %1 L-glutamin, %1 antibiyotik/antimycotic (100x) ve %0,1 glia türevi nörotrofik faktör (GDNF) ile nörobakal A ortamını oluşturan nöron media A'yı hazırlayın. 9,5 mL nörobasal A ortamına 200 μL B-27, 100 μL FBS, 100 μL L-glutamin, 100 μL antibiyotik/antimycotic (100x) ve 10 μL GDNF (10 μg/mL) ekleyin. NOT: B-27, L-glutamin ve GDNF'nin tazeliğini sağlamak için kullanımdan sonraki bir hafta içinde nöron media A'yı hazırlayın.</li>
	<li>Nöron media A'nın hazırlanmasıyla aynı prosedürü kullanarak ancak FBS eklemeden B nöron medyasını hazırlayın.</li>
	<li>10 mL oksijen stabil Krebs çözeltisinde 20 mg kollajenaz tip II, 6 mg sığır serum albümini ve 200 μL antibiyotik/ antimikkotik (100x) eriterek sindirim çözeltisi 1'i hazırlayın.</li>
	<li>Hank'in dengeli tuz çözeltisinin 4 mL'si ile %0,25 tripsin 1 mL seyrelterek sindirim çözeltisi 2 hazırlayın.</li>
	<li>Kaplamalı kapaklı bir tabak hazırlayın.<ol>
<li>Cam kapakları 48 kuyulu bir kültür plakasına yerleştirirken laminer akış tezgahında uzunlamasına kullanın.</li>
		<li>Poli-D-lizin stoku olarak0,1mg/mL konsantrasyonda ddH 2 O ile seyreltin. Stoku –20 °C'de saklayın ve kullanmadan önce çözün.</li>
		<li>Her kapak kapağının üzerine 40 μL poli-D-lizin stoğu ekleyin ve çözeltiyi oda sıcaklığında 10 dakika kuluçkaya yatırın. NOT: Farklı bazal alanlara sahip farklı plakalar için konsantrasyonu 4 μg/cm2olarak ayarlayın. Örneğin, 24 kuyulu bir plakadan bir kuyunun bazal alanı 2 cm2ise, 80 μL poli-D-lizin stoğunu bir kuyuya aktarmalıyız. Her cm2 4 μg poli-D-lizin içerir.</li>
		<li>48 kuyulu plakadaki poli-D-lizini çıkarın ve kapak örtülerini ddH2O üç kez durulayın.</li>
		<li>Plakanın susuz olduğundan emin olmak için plakayı laminer akış davlumbazında en az 30 dakika kurutun.</li>
		<li>Plakayı laminin kaplamadan önce en fazla 1 gün boyunca 4 °C'de saklayın. Plakanın −20 °C'de saklanması uzun süreli depolama için tercih edilir.</li>
		<li>Laminin'i 4 °C'de çözün. Laminin stok olarak 50 μg / mL konsantrasyonda ddH2O ile seyreltin. Stoku –20 °C'de saklayın ve kullanmadan önce 4 °C'de çözün.</li>
		<li>100 μL seyreltilmiş laminin'i her kapak kapağının üstüne aktarmak ve 1 saat boyunca 4 °C'de laminin kuluçkaya yatırmak için bir pipet kullanın. NOT: Farklı bazal alanlara sahip farklı plakalar için konsantrasyonu 5 μg/cm2olarak ayarlayın. Örneğin, 24 kuyulu bir plakadan bir kuyunun bazal alanı 2 cm2ise, bir kuyuya 200 μL seyreltilmiş laminin aktarın. Her cm2 5 μg laminin içerir.</li>
		<li>Laminin çözeltisini çıkarın ve kapak örtülerini bir kez ddH2O ile durulayın. Kazıma önlemek için bu işlemi kapak kapağının kenarında gerçekleştirin. NOT: Kaplamalı kapaklar en fazla 2 hafta boyunca 4 °C'de saklanabilir.</li>
	</ol>
</li>
</ol>2. Mesane hasadı
<ol><li>Beş haftalık Sprague-Dawley fareleri elde edin.</li>
	<li>Karbojeni (%95 oksijen,%5 CO 2)krebs çözeltisine bir buz banyosunda en az 30 dakika süreyle demlenerek sabit bir oksijen durumuna ve pH seviyesine ulaşın.</li>
	<li>Servikal çıkık yoluyla ötanaziden sonra, sıçanları sterilizasyon için 30 sn boyunca% 75 etanol içine batırın.</li>
	<li>Sıçanları sterilize edilmiş bir cerrahi havluya yerleştirin ve karınlarını açığa çıkar. Karın boşluğunu açın ve mesaneyi bir makas ve ön ayak seti ile ortaya dökün.</li>
	<li>Mesaneyi nazikçe kaldırın ve çapraz kontaminasyonu önlemek için mesane boynunu başka bir makas ve tostiki seti ile mesane boynundan kesin. Hücre sağkalımını iyileştirmek için mesaneyi hızlı bir şekilde soğuk oksijene stabil Krebs çözeltisine yerleştirin. NOT: Mesane çıkarıldıktan sonra, nöron sağkalım olasılığını iyileştirmek için aşağıdaki işlemleri hızlı bir şekilde gerçekleştirin.</li>
	<li>Krebs solüsyonlarını üç cam tabak ve cam kırıcıya ekleyin.</li>
	<li>Ön soğutma için buz banyosunda Krebs solüsyonu ile cam tabaklar ve cam kırıcılar hazırlayın.</li>
	<li>Karışıklığı önlemek için bu kapları 1–3 numaralarıyla işaretleyin.</li>
	<li>Her bir cam tabağı, bir kaşık çekirdeği bölücü ile eşleştirin.</li>
	<li>Cam tabak 1'de, mesaneyi oftalmik makasla kesin ve önps ve kaşık çekirdeği bölücü ile açın.</li>
	<li>Mesaneyi cam kırıcı 1'de durulayın ve cam tabağa yerleştirin 2.</li>
	<li>Cam tabaktaki tokmakları ve oftalmik makası kullanarak doku yüzeyindeki yapışan yağı ortadan kaldırın 2.</li>
	<li>Mesaneyi cam kırıcı 2'de durulayın ve cam tabağa yerleştirin 3.</li>
	<li>Eksojen ataşmanları çıkarmak için mesaneyi cam tabak 3'e kanatçıklar ve kaşık çekirdek bölücü kullanarak hafifçe kazıyın.</li>
	<li>Mesaneyi cam kırıcı 3'te durulayın, mesaneyi 14 mL soğuk Krebs çözeltisine sahip 15 mL santrifüj tüpüne aktarın ve numuneyi 356 x g ve 4 °C'de 1 dakika döndürün.</li>
	<li>Kontaminasyonu azaltmak için Krebs çözeltisi ile diğer iki tüpte önceki adımı iki kez tekrarlayın.</li>
</ol>3. İki aşamalı mesane sindirimi
<ol><li>Mesaneyi santrifüj tüpünden 1 mL sindirim çözeltisi içeren 2 mL şişeye aktarın 1. Mesaneyi çözelti halinde küçük parçalara (1 mm'den küçük) kesmek için oftalmik makas kullanın.</li>
	<li>Mesane çözeltisini steril bir hücre kültürü çanağına (100 mm çapında) 9 mL sindirim çözeltisi 1 ile karıştırın. %5 CO2,37 °C ve 200 rpm altında 1 saat boyunca sallanan bir inkübatörde 1.</li>
	<li>1. adım sindirimden sonra, çözeltiyi 8 dakika boyunca 4 °C'de 356 x g'da santrifüj edin.</li>
	<li>Sindirim çözeltisi 2'yi ön ısıtma için 37 °C su banyosuna yerleştirin.</li>
	<li>Santrifüjlemeden sonra, sindirim çözeltisi 1 içeren üstnatantı çıkarın ve hücre tortusunu hasat edin. Kalan bazı sıvılara izin verilir. Çözeltinin tamamen çıkarılması hücre kaybını artırabilir.</li>
	<li>Hücre tortusunu 15 mL santrifüj tüpünde ılık sindirim çözeltisi 2 ile karıştırın ve 37 °C su banyosunda 5 dakika sindirirken karışımı çalkalayın. 7 dakikayı aşmayın adım 2 sindirim veya nöronlar yok olacaktır.</li>
	<li>Sindirimden sonra, trypsin'i 10 mL soğuk durulama ortamı ile karışımda derhal devre dışı bırakın. NOT: Aşağıdaki adımları 0 °C–4 °C'de gerçekleştirin. Bir buz banyosu böyle bir durum sağlayabilir.</li>
	<li>Santrifüjlemeden sonra hücre tortusunu 8 dakika boyunca 4 °C'de 356 x g'da hasat edin. Kalan tripsin hücre büyümesine zararlı olduğundan mümkün olduğunca fazla medya çıkarın.</li>
	<li>Tortuları 3 mL nöron ortamı ile nazikçe yeniden sunun. Yüksek sağkalım oranı için hücre içeren çözeltide hava kabarcıklarının oluşmamasını sağlayın.</li>
	<li>Karışım medyasını 70 μm hücre süzgeçten 50 mL santrifüj tüpüne filtreleyin.</li>
	<li>Filtrat 30 dakika boyunca bir buz banyosunda 30 rpm'de bir çalkalayıcı üzerinde tutun. Bu adım gerekli değildir, ancak önerilir.</li>
	<li>Hücreleri 8 dakika boyunca 4 °C'de 356 x g'da santrifüjleme ile toplayın ve hücre peletlerini 1 mL nöron ortamı A'da hafifçe yeniden biriktirin.</li>
	<li>Hazırlanan 48 kuyu plakasının her kuyusuna 500 μL hücre karışımı ekleyin.</li>
	<li>37 °C ve %5 CO2'debir inkübatörde kültür hücreleri.</li>
	<li>Serumsuz bir kültür sağlamak için tüm medyayı 1 saat içinde nöron media B ile değiştirin.</li>
	<li>Nöron media B'nin yarısını her 3 günde bir değiştirin. NOT: Nöronlar 5-7 günlük kültürden sonra immünosimyasal deneylere hazırdır.</li>
</ol>

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Yazarlar büyük bir çıkar çatışması olmadığını beyanlarlar.

Plaka Hazırlama İmmünofluoresan veya kalsiyum görüntüleme deneyleri için 6, 12 veya 48 kuyulu kültür plakalarında cam kapakların kullanılması ekonomik ve örneklemsiz bir işlemdir. Hücreler, birincil hücre kültürlerinin hazırlanması sırasında kapaksız plakalarda iyi büyür. Bu nedenle, kapaklar Batı blot veya polimeraz zincir reaksiyonu gibi deneylerde dağıtılabilir. Ayrıca, kaplama, kapaklı veya kapaksız hücreleri kaplamadan önce gerekli bir adımdır. Laminin ve poli...

Alt idrar yollarının iki ana işlevi vardır: periyodik idrar depolama ve miktürasyon1. Alt idrar yolu sinir sistemi (LUTNS) bu fonksiyonları kontrol eder ve hassas ve birçok nöropatiye duyarlıdır, doğuştan gelen (porfiri), edinilmiş (Lyme hastalığı), hastalık durumlarına ikincil (diyabetik sistopati), ilaca bağlı (hemorajik sistit), neden olduğu ameliyat (abdominoperineal rezeksiyon) veya yaralanma (travmatik omurilik yaralanması)2, 3,4,5,6,7olabilir. Fizyolojik/patolojik çalışmalarda in vivo ve in vitro deneyler de aynı derecede önemlidir. LUTNS ile ilgili in vivo araştırmalar bir süredir organ, hücresel ve moleküler düzeylerde yapılırken, idrar kesesinden birincil nöronlar üzerinde in vitro araştırmalar yok denecek kadarazdır 8,9. Mevcut çalışma sınırlı olsa da, diğer araştırmacıların geliştirebilmesi için bu alandaki araştırmalara öncülük etmeyi umuyoruz. Bu şekilde, bu ortak kültür, mesane nöron disfonksiyonu gibi fenotiplerdeki fizyolojik disfonksiyonun hücresel olarak anlaşılmasına yol açabilir.
Kas hücrelerinin ayrık katmanlara açık bir yönüne sahip enterik kasların aksine, mesanenin kasları örgütsüz10. Bu nedenle, bu yöntem mesanenin dış tabakasını soymak yerine, çalışma zorlığını azaltmak ve yüksek hücre sağkalım oranı için ön işlem süresini kısaltmak için tüm mesaneyi sindirmeyi önerir.
Bu yöntemi izleyerek, nöronların ve diğer hücrelerin karışık bir kültürünü elde edebiliriz. Diğer hücreler vazgeçilmezdir, çünkü varlıkları bir in vivo ortamı taklit eder11. Ek olarak, bu tür hücreler ortamda bulunmayan maddeleri sağlar.
Bu yöntem sindirim için iki adım içerir. İlk olarak, kollajen tip II, kollajeni hidroliz yapmak için kullanılır, ardından tripsin, dokuyu hücrelere dağıtmak için10. Bu şekilde, mesane dokuları tek hücrelere dağılır ve daha sonra nispeten bağımsız büyür. Nöronların kültürü olgunlaştığında, nöronlar görüntüleme veya fonksiyonel tahliller için kullanılabilir.

<table><tbody><tr><td>0.25% trypsin</td><td>Gibico</td><td>15050065</td><td>Enzyme digestion</td></tr><tr><td>48-well culture plate</td><td>Corning</td><td>3548</td><td>Coating dish</td></tr><tr><td>antibiotic/antimycotic</td><td>Gibico</td><td>15240062</td><td>Culture media/Rinse media</td></tr><tr><td>Anti-Glial Fibrillary Acidic Protein Antibody</td><td>Santa Cruz</td><td>sc-33673</td><td>ICC</td></tr><tr><td>B-27</td><td>Gibico</td><td>17504044</td><td>Culture media</td></tr><tr><td>BSA Fraction V</td><td>Gibico</td><td>332</td><td>Enzyme digestion</td></tr><tr><td>Choline Acetyltransferase Antibody</td><td>Abcam</td><td>ab18736</td><td>ICC</td></tr><tr><td>CO2 Incubator</td><td>Heraeus</td><td>B16UU</td><td>Cells culture</td></tr><tr><td>Collagenase type II</td><td>Sigma</td><td>2593923</td><td>Enzyme digestion</td></tr><tr><td>DMEM/F-12</td><td>Gibico</td><td>11330032</td><td>Rinse media</td></tr><tr><td>DYNLL2 Antibody</td><td>Santa Cruz</td><td>sc-13969</td><td>ICC</td></tr><tr><td>Fetal Bovine Serum</td><td>Gibico</td><td>10100147</td><td>Culture media/Rinse media</td></tr><tr><td>Forceps</td><td>Shanghai Jin Zhong Medical Devices</td><td>1383</td><td>10 cm; Sterile operation</td></tr><tr><td>Glass breakers</td><td>Huan Qiu Medical Devices</td><td>1101</td><td>50 ml; Sterile operation</td></tr><tr><td>Glass coverslips</td><td>WHB Scientific</td><td>WHB-48-CS</td><td>Coating dish</td></tr><tr><td>Glass dishes</td><td>Huan Qiu Medical Devices</td><td>1177</td><td>100 mm; Sterile operation</td></tr><tr><td>Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 488</td><td>Southernbiotech</td><td>3050-30</td><td>ICC</td></tr><tr><td>Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 555</td><td>Southernbiotech</td><td>3050-30</td><td>ICC</td></tr><tr><td>Hoechst 33342</td><td>BD</td><td>561908</td><td>ICC</td></tr><tr><td>Laminar flow bench</td><td>Su Jie Medical Devices</td><td>CB 1400V</td><td>Sterile operation</td></tr><tr><td>Laminin</td><td>Sigma</td><td>L2020</td><td>Coating dish</td></tr><tr><td>L-glutamine</td><td>Gibico</td><td>25030081</td><td>Culture media</td></tr><tr><td>MAP-2 Antibody</td><td>Affinity</td><td>AF5156</td><td>ICC</td></tr><tr><td>Murine GDNF</td><td>Peprotech</td><td>AF45044</td><td>Culture media</td></tr><tr><td>Neurobasal-A Medium</td><td>Gibico</td><td>10888022</td><td>Culture media</td></tr><tr><td>Ophthalmic scissors</td><td>Shanghai Jin Zhong Medical Devices</td><td>J21010</td><td>12.5 cm; Sterile operation</td></tr><tr><td>Pipettes</td><td>Eppendorf</td><td>3120000240</td><td>100-1000 ul; Reagent and sample pipetting</td></tr><tr><td>Pipettes</td><td>Eppendorf</td><td>3120000267</td><td>10-100 ul; Reagent and sample pipetting</td></tr><tr><td>Poly-D-lysine</td><td>Sigma</td><td>P7280</td><td>Coating dish</td></tr><tr><td>Refrigerated centrifuge</td><td>Ping Fan Instrument</td><td>TGL-16A</td><td>Enzyme digestion</td></tr><tr><td>Shaking incubator</td><td>Haimen Kylin-Bell Lab Instruments</td><td>T8-1</td><td>Enzyme digestion</td></tr><tr><td>SLC17A9 Antibody</td><td>MBL International</td><td>BMP079</td><td>ICC</td></tr><tr><td>Spoons nucleus divider</td><td>Shanghai Jin Zhong Medical Devices</td><td>YZR030</td><td>12 cm; Sterile operation</td></tr><tr><td>Substance P Antibody</td><td>Santa Cruz</td><td>sc-58591</td><td>ICC</td></tr><tr><td>Surgical scissors</td><td>Shanghai Jin Zhong Medical Devices</td><td>J21130</td><td>16 cm; Sterile operation</td></tr><tr><td>Surgical towel</td><td>Fu Kang Medical Devices</td><td>5002</td><td>40 x 50 cm; Sterile operation</td></tr><tr><td>Synapsin-1 Antibody</td><td>CST</td><td>5297T</td><td>ICC</td></tr><tr><td>Tubulin beta Antibody(&amp;beta;-III-tubulin)</td><td>Affinity</td><td>AF7011</td><td>ICC</td></tr></tbody></table>

isolation and culture of primary neurons and glia from adult rat urinary bladder

Alt idrar yolu, periyodik idrar depolama ve miktürasyon olmak üzere iki ana işleve sahiptir; bu fonksiyonlar merkezi ve periferik nöroregülasyon yoluyla aracılık edilir. Alt idrar yolu sinir sistemi hakkında kapsamlı araştırmalar yapılmış olmasına rağmen, çoğu çalışma birincil kültüre odaklanmıştır. Bu protokol, Sprague-Dawley sıçanlarından mesane nöronlarının ve glialarının izolasyonu ve kültürü için bir yöntem sunar. Bu yöntemde nöronlar ve glialar 5-7 gün boyunca 37 °C,%5 CO 2 inkübatörde inkübe edildi. Sonuç olarak, ilgili sonraki immünofluoresans deneyleri için uygun olgun şekillere dönüştüler. Hücreler morfolojik olarak optik mikroskop kullanılarak gözlendi. Nöronlar, sinaptik veziküller ve glia sırasıyla β-III-tubulin ve MAP-2, Synapsin-1 ve GFAP boyama ile tanımlanmıştır. Bu arada, immünosistokimya kolin asetiltransfenaz, DYNLL2 ve SLC17A9 gibi nörotransmitter ile ilgili çeşitli proteinler üzerinde yapıldı.

Birincil hücre kültürü sürecinde, elde edilen hücreler ekli durumdan önce parlak ve net sınırlarla yuvarlaktı. Nöronlar büyüdükçe dendritler ve aksonlar farklı olmaya başladı. 5-7 günlük kültürden sonra, nöronlar görüntüleme veya fonksiyon çalışmaları için ideal olan uzun projeksiyonlarla olgun bir forma ulaştılar. Değişen ortam nedeniyle safsızlıkların ve hücre kalıntılarının çoğu giderilebilse de poli-D lizin ve laminin kaplamaya bağlı bazı kalıntılar görülebiliyordu<...

Watch this Scientific Journal Video about Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder at JoVE.com

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

Bu protokol, birincil nöronlar için tekrarlanabilir bir protokol oluşturmaya ve daha fazla hücresel deney için sıçan mesanesinden glia izolasyonu oluşturmaya çalışır.

Yetişkin Sıçan İdrar Kesesinden Primer Nöronların ve Gliaların İzolasyonu ve Kültürü

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

isolation-culture-primary-neurons-glia-from-adult-rat-urinary

Research

JoVE Journal

Neuroscience

3.8K Görüntüleme.  Guangzhou University of Chinese Medicine. Bu protokol, birincil nöronlar için tekrarlanabilir bir protokol oluşturmaya ve daha fazla hücresel deney için sıçan mesanesinden glia izolasyonu oluşturmaya çalışır.

Video: Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Periventriküler Yetişkin Rat Bölgesinde ve İnsan Omurilik gelen Nöral Kök / Progenitör Hücreler izolasyonu

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Gelişim Biyolojisi

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

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 ...

İmmünoloji ve Enfeksiyon

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

İlköğretim Rat Kortikal Nöronlar ve Glia bilaminar Co-kültür

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Nörobilim

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

Sıçan Embriyonik Nöral Hücre İzolasyonu ve Kültür: Bir Hızlı Protokol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Embriyonik Kemirgen Brain itibaren İlköğretim Mesensefalik dopaminerjik Nöronlar İzolasyonu, Kültür ve Uzun Vadeli Bakım

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

Yetişkin Fare Omurilik Doku İlköğretim Karışık glial Kültürler hazırlanması

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

Dorsal kök gangliyon yalıtım ve birincil kültür nörotransmitter yayın çalışmaya

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.

Yetişkin İnsan Beyin Dokusundan İnsan Mikroglia elde

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

Isolation and Culture of Primary Neurons and Glia from a Rat Urinary Bladder

Source: Wang, R. et al., <a href="https://app.jove.com/v/61177">Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder.</a> J. Vis. Exp. (2020).
This video demonstrates a technique for generating a primary culture of neurons and glia from a rat&#39;s urinary bladder. It outlines the steps involved in tissue preparation, enzymatic digestion to obtain single cells, and culturing the cells in a neuron-specific medium to establish a primary neuronal and glial culture.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Nöronal Kültür Teknikleri

Birincil Nöronal Kültür

Isolation of Neural Stem Progenitor Cells from the Spinal Cord Periventricular Region of an Adult Rat

Source: Mothe, A., et al., <a href="https://app.jove.com/v/52732">Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord.</a> J. Vis. Exp. (2015).
This video presents a method for isolating neural stem/progenitor cells from the periventricular tissue of the spinal cord in adult rats. The technique includes incubating the tissue fragments in a solution containing proteolytic enzymes to disrupt the extracellular matrix and release the cells. Subsequently, the released cells are plated in a medium containing specific growth factors that enhance neural stem/progenitor proliferation and promote aggregation into three-dimensional neurospheres.

Erişkin Bir Sıçanın Omurilik Periventriküler Bölgesinden Nöral Kök Progenitör Hücrelerin İzolasyonu

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Preparation of Dissection ...