Name: isolation and culture of primary neurons and glia from adult rat urinary bladder
Uploaded: 2020-05-23T14:45:00
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 ...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" style="width:1061px;" width="1061">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">0.25% trypsin</td>
			<td style="width:279px;">Gibico</td>
			<td style="width:119px;">15050065</td>
			<td style="width:296px;">Enzyme digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">48-well culture plate</td>
			<td>Corning</td>
			<td style="width:119px;">3548</td>
			<td>Coating dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">antibiotic/antimycotic</td>
			<td>Gibico</td>
			<td>15240062</td>
			<td>Culture media/Rinse media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Glial Fibrillary Acidic Protein Antibody</td>
			<td>Santa Cruz</td>
			<td>sc-33673</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B-27</td>
			<td>Gibico</td>
			<td>17504044</td>
			<td>Culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA Fraction V</td>
			<td>Gibico</td>
			<td>332</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Choline Acetyltransferase Antibody</td>
			<td>Abcam</td>
			<td>ab18736</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CO2 Incubator</td>
			<td>Heraeus</td>
			<td>B16UU</td>
			<td>Cells culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase type II</td>
			<td>Sigma</td>
			<td>2593923</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">DMEM/F-12</td>
			<td>Gibico</td>
			<td>11330032</td>
			<td>Rinse media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DYNLL2 Antibody</td>
			<td>Santa Cruz</td>
			<td>sc-13969</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Gibico</td>
			<td>10100147</td>
			<td>Culture media/Rinse media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps</td>
			<td>Shanghai Jin Zhong Medical Devices</td>
			<td>1383</td>
			<td>10 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass breakers</td>
			<td>Huan Qiu Medical Devices</td>
			<td>1101</td>
			<td>50 ml; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass coverslips</td>
			<td>WHB Scientific</td>
			<td>WHB-48-CS</td>
			<td>Coating dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass dishes</td>
			<td>Huan Qiu Medical Devices</td>
			<td>1177</td>
			<td>100 mm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 488</td>
			<td>Southernbiotech</td>
			<td>3050-30</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 555</td>
			<td>Southernbiotech</td>
			<td>3050-30</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342</td>
			<td>BD</td>
			<td>561908</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminar flow bench</td>
			<td>Su Jie Medical Devices</td>
			<td>CB 1400V</td>
			<td>Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminin</td>
			<td>Sigma</td>
			<td>L2020</td>
			<td>Coating dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine</td>
			<td>Gibico</td>
			<td>25030081</td>
			<td>Culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MAP-2 Antibody</td>
			<td>Affinity</td>
			<td>AF5156</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Murine GDNF</td>
			<td>Peprotech</td>
			<td>AF45044</td>
			<td>Culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neurobasal-A Medium</td>
			<td>Gibico</td>
			<td>10888022</td>
			<td>Culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ophthalmic scissors</td>
			<td>Shanghai Jin Zhong Medical Devices</td>
			<td>J21010</td>
			<td>12.5 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipettes</td>
			<td>Eppendorf</td>
			<td style="width:119px;">3120000240</td>
			<td>100-1000 ul; Reagent and sample pipetting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipettes</td>
			<td>Eppendorf</td>
			<td style="width:119px;">3120000267</td>
			<td>10-100 ul; Reagent and sample pipetting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-D-lysine</td>
			<td>Sigma</td>
			<td>P7280</td>
			<td>Coating dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Refrigerated centrifuge</td>
			<td>Ping Fan Instrument</td>
			<td>TGL-16A</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shaking incubator</td>
			<td>Haimen Kylin-Bell Lab Instruments</td>
			<td style="width:119px;">T8-1</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SLC17A9 Antibody</td>
			<td>MBL International</td>
			<td>BMP079</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spoons nucleus divider</td>
			<td>Shanghai Jin Zhong Medical Devices</td>
			<td>YZR030</td>
			<td>12 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Substance P Antibody</td>
			<td>Santa Cruz</td>
			<td>sc-58591</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical scissors</td>
			<td>Shanghai Jin Zhong Medical Devices</td>
			<td>J21130</td>
			<td>16 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical towel</td>
			<td>Fu Kang Medical Devices</td>
			<td>5002</td>
			<td>40 x 50 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Synapsin-1 Antibody</td>
			<td>CST</td>
			<td>5297T</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tubulin beta Antibody(&#946;-III-tubulin)</td>
			<td>Affinity</td>
			<td>AF7011</td>
			<td>ICC</td>
		</tr>
	</tbody>
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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 ...

Research

JoVE Journal

Neuroscience

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Embriyonik Fare Hipokampal ve Kortikal nöronlar primer Nucleofection ve Kültür

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

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 ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Prenatal Fareler gelen Hipokampal Nöronlar izolasyonu ve Kültürü

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

Bir<em&gt; In-vitro</emYetişkin Mouse myenterik Pleksus izole edilen enterik Nöronlar ve Glia bir&gt; hazırlanması

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

Primary Culture of Mouse Dopaminergic Neurons

Fare dopaminerjik nöronlar İlköğretim Kültür

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Civciv Embriyolar itibaren İzolasyon ve Disosiye Duyu Nöronlar Kültür

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

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

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 ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Fare Subcallosal Zone İzolasyon ve Yetişkin Nöral Kök Hücre Kültürü

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

Yenidoğan Faresinden İzole Edilen Spinal Kord Nöronları İzolasyonu ve Kültürü

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

E14-E16 Sprague Dawley Sıçan Embriyosu İzole Karışık Primer Hipokampal ve Kortikal Nöronlardan Nörosfer Lerin Üretimi

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...