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>

<ol>
	<li>Fowler, C. J., Griffiths, D., de Groat, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neural+control+of+micturition.">The neural control of micturition.</a> Nature Reviews Neuroscience. 9 (6), 453-466 (2008).</li><li>Golbidi, S., Laher, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bladder+dysfunction+in+diabetes+mellitus.">Bladder dysfunction in diabetes mellitus.</a> Frontiers in Pharmacology. 1, 136 (2010).</li><li>Iwasawa, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+Effects+of+Intravenous+Cyclophosphamide+in+Combination+with+Mesna+Provided+Intravenously+and+via+Bladder+Perfusion+in+a+Patient+with+Severe+Multifocal+Motor+Neuropathy.">Long-term Effects of Intravenous Cyclophosphamide in Combination with Mesna Provided Intravenously and via Bladder Perfusion in a Patient with Severe Multifocal Motor Neuropathy.</a> Internal Medicine. 56 (14), 1893-1896 (2017).</li><li>Lange, M. M., van de Velde, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urinary+and+sexual+dysfunction+after+rectal+cancer+treatment.">Urinary and sexual dysfunction after rectal cancer treatment.</a> Nature Reviews. Urology. 8 (1), 51-57 (2011).</li><li>Lin, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+function+and+dysfunction+in+acute+intermittent+porphyria.">Nerve function and dysfunction in acute intermittent porphyria.</a> Brain. 131, 2510-2519 (2008).</li><li>Rantell, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+the+utility+of+urodynamics,+including+ambulatory,+and+24+h+monitoring,+in+predicting+upper+urinary+tract+damage+in+neuro-urological+patients+and+other+lower+urinary+tract+dysfunction?+ICI-RS+2017.">What is the utility of urodynamics, including ambulatory, and 24 h monitoring, in predicting upper urinary tract damage in neuro-urological patients and other lower urinary tract dysfunction? ICI-RS 2017.</a> Neurourology and Urodynamics. 37, 25-31 (2018).</li><li>Halperin, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+management+of+Lyme+neuroborreliosis.">Diagnosis and management of Lyme neuroborreliosis.</a> Expert Review of Anti-Infective Therapy. 16 (1), 5-11 (2018).</li><li>Walter, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+of+supraspinal+correlates+to+lower+urinary+tract+stimulation+in+healthy+participants+-+A+fMRI+study.">Reliability of supraspinal correlates to lower urinary tract stimulation in healthy participants - A fMRI study.</a> Neuroimage. 191, 481-492 (2019).</li><li>Leitner, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+infusion-drainage+device+to+assess+lower+urinary+tract+function+in+neuro-imaging.">A novel infusion-drainage device to assess lower urinary tract function in neuro-imaging.</a> BJU International. 119 (2), 305-316 (2017).</li><li>Smith, T. H., Ngwainmbi, J., Grider, J. R., Dewey, W. L., Akbarali, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.</a> Journal of Visualized Experiments. (78), e50688 (2013).</li><li>Gordon, J., Amini, S., White, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+overview+of+neuronal+cell+culture.">General overview of neuronal cell culture.</a> Methods in Molecular Biology. 1078, 1-8 (2013).</li><li>Roppongi, R. T., Champagne-Jorgensen, K. P., Siddiqui, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-Density+Primary+Hippocampal+Neuron+Culture.">Low-Density Primary Hippocampal Neuron Culture.</a> Journal of Visualized Experiments. (122), e55000 (2017).</li><li>Arms, L., Vizzard, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropeptides+in+lower+urinary+tract+function.">Neuropeptides in lower urinary tract function.</a> Handbook of Experimental Pharmacology. (202), 395-423 (2011).</li><li>Moriyama, Y., Hiasa, M., Sakamoto, S., Omote, H., Nomura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+nucleotide+transporter+(VNUT):+appearance+of+an+actress+on+the+stage+of+purinergic+signaling.">Vesicular nucleotide transporter (VNUT): appearance of an actress on the stage of purinergic signaling.</a> Purinergic Signal. 13 (3), 387-404 (2017).</li><li>Chaudhury, A., He, X. D., Goyal, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+Va+plays+a+key+role+in+nitrergic+neurotransmission+by+transporting+nNOS&#945;+to+enteric+varicosity+membrane.">Myosin Va plays a key role in nitrergic neurotransmission by transporting nNOS&#945; to enteric varicosity membrane.</a> American journal of physiology. Gastrointestinal and Liver Physiology. 301 (3), 498-507 (2011).</li><li>Le Berre-Scoul, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+enteric+neuron-glia+coculture+system+reveals+the+role+of+glia+in+neuronal+development.">A novel enteric neuron-glia coculture system reveals the role of glia in neuronal development.</a> The Journal of Physiology. 595 (2), 583-598 (2017).</li><li>Hayashi, H., Yamada, M., Kumai, J., Takagi, N., Nomizu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+activities+of+laminin-111-derived+peptide-chitosan+matrices+in+a+primary+culture+of+rat+cortical+neurons.">Biological activities of laminin-111-derived peptide-chitosan matrices in a primary culture of rat cortical neurons.</a> Archives of Biochemistry and Biophysics. 648, 53-59 (2018).</li><li>Bottenstein, J. E., Sato, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+a+rat+neuroblastoma+cell+line+in+serum-free+supplemented+medium.">Growth of a rat neuroblastoma cell line in serum-free supplemented medium.</a> Proceedings of the National Academy of Sciences of the United States of America. 76 (1), 514-517 (1979).</li><li>Brewer, G. J., Torricelli, J. R., Evege, E. K., Price, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+Survival+of+Hippocampal+Neurons+in+B27-Supplemented+Neurobasalm,+a+New+Serum-free+Medium+Combination.">Optimized Survival of Hippocampal Neurons in B27-Supplemented Neurobasalm, a New Serum-free Medium Combination.</a> Journal of Neuroscience Research. 35 (5), 567-576 (1993).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nature Protocols. 1 (5), 2406-2415 (2006).</li><li>Georgas, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+illustrated+anatomical+ontology+of+the+developing+mouse+lower+urogenital+tract.">An illustrated anatomical ontology of the developing mouse lower urogenital tract.</a> Development. 142 (10), 1893-1908 (2015).</li><li>Adam, M., Potter, A. S., Potter, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychrophilic+proteases+dramatically+reduce+single-cell+RNA-seq+artifacts:+a+molecular+atlas+of+kidney+development.">Psychrophilic proteases dramatically reduce single-cell RNA-seq artifacts: a molecular atlas of kidney development.</a> Development. 144 (19), 3625-3632 (2017).</li></ol>

Yazarlar büyük bir çıkar çatışması olmadığını beyanlarlar.

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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">
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		<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>
</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

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

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 ...

Nörobilim

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 ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

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

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

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

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

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

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

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

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

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

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 ...

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

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

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

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 postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

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 ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

Embriyonik Fare Beyincisinden İzole Edilen Nöronların Birincil Kültürü

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...