Biz editoryal yardım için Jonna Ellis teşekkür ederim. Ruh Sağlığı Ulusal Enstitüsü: açıklanan proje Ödülü Numarası R01MH079751 (F. Peruzzi PI) tarafından desteklenmiştir. Içeriği sadece yazarların sorumluluğundadır ve mutlaka Ruh Sağlığı Ulusal Enstitüsü veya Ulusal Sağlık Enstitüleri resmi görüşlerini temsil etmez.

1. Poli-D-Lizin (PDL): Hazırlanması <ol><li> 1 mg / ml &#39;lik bir stok solüsyonu elde etmek için PDL 5 mg steril GKD 2 O 5 ml ilave edilir. </li><li> Birkaç kez pipetleme tarafından stok solüsyonu karıştırın. </li><li> Hemen kullanın veya 2-8 Poly-D-Lizin Çözüm saklamak ° C </li></ol> 2. Poly-D-Lizin (PDL): Kaplama Plastik Hücre Kültürü Yemekleri <ol><li> Steril GKD 2 O ile 10 ug / ml &#39;lik nihai konsantrasyona PDL stok solüsyonu ile seyreltilir. </li><li> Kültür yüzey alanı (60 mm çanak için 3 ml) kapsayacak şekilde 60 mm çanak pipetleyin yeterli bir çözüm. </li><li> Kültür yüzey hatta kaplama sağlamak için hafifçe sallayın. </li><li> Gece boyunca oda sıcaklığında (RT) az kaplı inkübe edin. </li><li> Ertesi gün, genellikle diseksiyon gün, aspirasyon tarafından Poly-D-Lizin Çözüm kaldırmak ve steril GKD 2 O 3 ml kısaca yıkayın Bu adımı yineleyin. İkinci yıkamadan sonra, aspirasyon tamamen su çıkarmak. </li><li>Plakalar üç hafta için 4 ° C&#39;de saklanabilir. </li></ol> 3. Poly-D-Lizin (PDL) ve Laminin: Cam hazırlanması ve Kaplama İki odacıklı Slaytlar <ol><li> Sırasıyla, 10 ve 5 ug / ml, nihai konsantrasyon Steril GKD 2 O karışımı PDL (1 mg / ml) ve laminin (1 mg / ml) stok çözeltileri. </li><li> Cam bir kuyu iki odacıklı slayt kültür yüzey alanı (2 hatta cam çift odacıklı slayt her iyi için 1 ml) kapsayacak şekilde pipetleyin yeterli bir çözüm. </li><li> Kültür yüzey hatta kaplama sağlamak için hafifçe sallayın. </li><li> Gece boyunca oda sıcaklığında kaplı inkübe edin. </li><li> Ertesi gün, aspirasyon yoluyla Poly-D-Lizin-Laminin kaplama çözümü kaldırmak ve steril GKD 2 O. 1 ml ile iki kez kısaca yıkayın İkinci yıkamadan sonra, aspirasyon tamamen su çıkarmak. </li><li> Odacık slaytlarınızı üç hafta için 4 ° C&#39;de saklanabilir. </li></ol> Not: Herhangi bir cam haznesi slayt izlemeye kaplanabilirg bu protokol. Her bir slayt kontrolü test Deneysel ortamda (örneğin tedavi edilmezse karşı tedavi, untransfected karşı transfekte) sağlar çünkü çoğu zaman çift odacıklı slaytlar kullanın. 4. Nöronal Diseksiyon ve Kültür <ol><li> 37 ° C su banyosunda aşağıdaki reaktifler Isınma: <ul type="disc"><li> Orijinal 100 ml şişe üzerine TrypLE Express. </li><li> Neurobasal/B27 tam orta (tablo görüyorum). Isıttı hacmi (on 60 mm kaplamalı yemekler örneğin 30 ml) kaplamalı olarak yemeklerinin sayısına bağlıdır. </li></ul></li><li> Dört adet 60 mm&#39;lik kültür kaplarına ve 15 ml BD Falcon yüksek netlik polipropilen konik tüpe 13 ml soğuk Hazırda E çözeltisinden 3 ml ekleyin. </li><li> Üç 100 mm&#39;lik kültür kaplarına her birine soğuk diseksiyon orta (tablo II, Dr Olimpia Meucci, kişisel iletişim bakın) 25-30 ml ekleyin. Orta büyük bir hacmi içeren bu plakaları, hemen sonra embriyo yıkamak için kullanılacaktıramniyotik kese (adımları 4.7 ve 4.8) kaldırılması. </li><li> Laboratuvar Hayvanları Humane Bakımı ve Kullanımı Halk Sağlığı Hizmetleri Politikası&#39;na uygun olarak ve kurumsal olarak onaylanmış hayvan bakım altında CO 2 tarafından bir E17 zamanlı gebe sıçan Euthanize ve protokolünü kullanır. </li><li> % 70 EtOH ile alt karın püskürtün ve rahim ve embriyoların açığa bir makas ile deri ve kas yoluyla mediale kesti. </li><li> Bütün fetuslarda çıkarın ve soğuk diseksiyon orta (25-30 ml, adım 4.3 &#39;e bakınız) aşırı içeren steril 100 mm tabağına yerleştirin. </li><li> Amniyotik kese makas küçük bir çifti kullanılarak embriyo kesin ve soğuk diseksiyon orta içeren ikinci 100 mm tabağına yerleştirin. </li><li> 5-10 saniye hafifçe eğerek 100 mm çanak ile oda sıcaklığında embriyoların yıkayın. Daha sonra, diseksiyon orta içeren üçüncü 100 mm çanak Durulanmış embryo transfer. Aşırı orta İki yıkama genellikle kan tüm izlerini silmek için yeterlidir. Bununla birlikte, eğer imalatıaçıklı, soğuk diseksiyon orta 25-30 ml içeren taze bir 100 mm çanak ile bir kez daha yıkayın. </li><li> Stereomikroskopta ve kavisli bir forseps kullanarak, deri ve kafatası çekerek her rat embriyo beyni ayıklayın. Soğuk Hazırda E. ile 60 mm yemekleri (genellikle, tabağı başına en fazla 5 beyinleri ile) buz üzerinde bu plakalar tutun birinde bütün beyin yerleştirin. </li><li> Seferinde bir çanak alın ve bir mikroskop altında, hemisfer ayırmak ve orta beyin ve meninks çıkarmadan serebral korteks izole. </li><li> İsteğe Bağlı: orta hat boyunca kesme beyinleri, hipokampi ayıklamak ve hipokampal nöronlar yalıtmak için aşağıdaki prosedürü izleyin. </li><li> Tüm diseksiyonlar tamamlanıncaya kadar buz üzerinde serebral korteks bırakın soğuk Hazırda E. 13 ml içeren 15 ml berrak konik tüp tüm diseke korteksleri toplayın. Kendi küçük boyutları nedeniyle, disseke hipokampi yerine 15 ml tüp bir 1.5 ml Eppendorf tüp toplanabilir. Ya da bu adım korteksleri az, eğer istenirsehipokampi 1 ml&#39;lik bir şişe içinde cryotube yerleştirilebilir hazırda E + 2 +% B27 gentamisin (50 ug / ml) 2-4 korteksleri veya şişe başına 2-4 hipokampi oranında + Fungizone (250 ng / ml). Beyin dokusu kadar bir hafta (daha sonraki dönemlerde henüz test edilen değil) için karanlıkta 4 ° C&#39;de saklanabilir. Gerektiğinde, Hazırda E içeren 15 ml tüp için beyin dokusu aktarmak ve daha sonra nöronlar yalıtmak için aşağıdaki protokolü takip ince pens kullanın. </li><li> Bir doku kültürü kaput tüp aktarın. Korteksleri tüpün dibe sonra dikkatlice süpernatant kaldırmak için izin verin. </li><li> , 15 ml konik tüp taze Hazırda E 13 ml ekleyin korteksleri tüpün dibe çöktüğünden izin ve dikkatli supernatant çıkarın. Bu adım 2 kez daha tekrarlayın ve son yıkamadan sonra, özenle tüm medya çıkarın. </li><li> Sıcak Tr; enzimatik 1-2 ml (hipokampi izolasyonu için daha az kullanın korteksleri sayısına bağlı olarak) ekleyerek serebral korteks sindirmekypLE Express. Parafilm ile tüp kap kapatır ve 10 dakika boyunca 37 ° C&#39;de su banyosu içinde tüp yüzer. </li><li> Kap açılmadan önce% 70 etanol ile tüp püskürtmek ve korteksleri borusunun alt kısmında yerleşir ve süpernatan kaldırmak için izin hazırda E. 10 ml ilave edilir. Bu adım TrypLE Express yıkanmaya üç kez tekrarlayın. Son adım olarak, dikkatle tüm medya çıkarın. </li><li> Yavaşça (4-5 kez) bir yangın-parlatılmış cam Pasteur (çapı yaklaşık 1 mm) kullanılarak Neurobasal/B27 tam medyum 2 ml korteksleri öğütmek. Kabarcıkları önlemek için dikkatli olun. </li><li> Çapında küçük bir steril cam Pasteur pipeti (yani çapı 1/2-3/4 mm pipet) ile başka bir 4-5 kez tekrarlayın. Bir Pasteur pipeti bu küçük kullanmayın veya hücreleri yok edecek. </li><li> Yerleşmek için (eğer varsa, genellikle çok az) doku kalan parçaları izin verin. </li><li> Doku parçaları yerleşmiş bırakarak, yeni bir 15-ml tüpüne üst tek bir hücre süspansiyonu transfer edin. KürkNeurobasal/B27 tam orta 10-12 ml hücre süspansiyonu kadar sulandırmak incele. </li><li> İyice karıştırın ve bir 1.5 ml Eppendorf tüp 50x Sayma Çözüm (Tablo III) 490 ul hücre süspansiyonu 10 ul ekleyerek sayım için hücre seyreltik. </li><li> 5.0 x 10 4 / cm 2 arasında derişimde PDL-kaplı levhalar üzerine plakası hücreleri. Nucleofection yapılmasını mümkün olmaması durumunda, daha yüksek bir konsantrasyonu (8-10 x 10 4 / cm 2) de hücrelerin kaplama önermektedir. </li><li> Yaklaşık 13 x 10 6 nöronların her E17 fetus türetilen Normal olarak, biz, Deneme başına 9-10 fetus incelemek. Daha fazla embriyoların gerekirse, tüm prosedürü iki saatten fazla sürecek olmadığından emin olun. </li><li> Eğer istenirse, 24 saat sonra izole, sitosin-β-D-arabinofuranoside (AraC) 10 uM glial proliferasyonu önlemek amacıyla, her bir çanak eklenebilir. Neurobasal/B27 orta glial proliferasyonu inhibe Bununla birlikte, bu adım gerekli değildirGibi üreticinin önerilerine (Invitrogen / Gibco). </li><li> Kesin zaman istenen türev aşaması bağlı olmasına rağmen, nöronlar, in vitro olarak 4-5 gün sonra deneyler için kullanılabilir. Biz hayatta önemli bir azalma (Şekil 1) olmadan 4 hafta için kültürlü nöronlar var. </li><li> Genişletilmiş kültür için, taze hazırlanmış Neurobasal/B27 tam orta her hafta kültür ortamı değiştirin. </li></ol> 5.. Temsilcisi Sonuçlar Cam haznesi slaytlar nöronlar üzerinde kültive immünsitokimya tabi tutulabilir. Şekil 1 kültüründe beş gün sonra sabit ve nöronal işlemleri gösteren anti-MAP-2 antikoru ile immunolabeled bir kortikal nöron tipik bir görüntüsü gösterilmektedir. Şekil 2 kültüründe 3 hafta sonra bir sıçan hipokampal nöron bir temsilcisi görüntüsünü gösterir. Tamamen farklılaşmış hücre nöronal morfoloji MAP-2 tarafından vurgulanır immunolabeling (MAP-2 nöronal belirteç, fare monoklonal antikor klonunun AP-20, Gene Tex, Irvine, CA), daha önce tarif edildiği gibi 1 standart bir prosedür izlenerek,. Görüntüleri EXi aqua kamera (Qimaging), motorlu Z ekseni ve SlideBook5 kazanım / Dekonvolüsyonun yazılımı (Akıllı Görüntüleme Yenilikler, Inc, Denver, CO) ile donatılmış Nikon Eclipse E400 dik floresans mikroskobu ile görüntülenmiştir. Her bir resmin üç boyutlu görüntüleri bir dizi birini iki boyutlu resme deconvoluted ve çözünürlüğü artırmak için yakın maksimal yoğunluğu sinyali kesme ayarlayarak çözüldü. Şekil 3 nöronal kültürlerin saflığı gösterir. Protein lizatları DIV7 sıçan nöronal kültürlerin (ctx) gelen ve insan glioblastoma (GBM) bir durumda elde edilmiştir. Beklenildiği gibi, nöronal lizat nöronal protein MAP-2 ve astrositik işaretleyici GFAP negatif için güçlü bir şekilde pozitif olduğu; GBM proteini lizat negatif f ikenya GFAP MAP-2 ve pozitif. Bizim protokol biz orta diseksiyon ve durulama olarak yıllarca Hazırda E kullanarak rağmen, son zamanlarda biz daha fazla kullanmak için beyin dokuları korumak için ek bir ve çok pratik kullanım incelemiş bulunuyoruz. Şekil 4 in vitro 5 (DIV5) bir gün göstermektedir embriyosu kendi orijinal Diseksiyon sonra hazırda E + B27 in bir hafta süreyle 4 ° C de muhafaza korteksleri izole sıçan kortikal nöronların kültürü. Nöronlar daha önce tarif edildiği gibi PDL ve laminin ile kaplanmış bir cam iki bölmeli kayar üzerine ekildi. Elde edilen görüntü SlideBook5 toplama / yukarıda tarif edildiği gibi Dekonvolüsyonun yazılımı (Şekil 2) kullanılarak deconvoluted edildi. <img alt="Şekil 1" src="/files/ftp_upload/3965/3965fig1.jpg" /> Şekil 1. PmaxGFP (Amaxa, Lonza, Walkersville, MD) ile nucleofected ve MAP ile immunolabeled bir kortikal nöron Temsilcisi görüntü-2 Antikor, kırmızı. Orijinal büyütme 100x. <img alt="Şekil 2" src="/files/ftp_upload/3965/3965fig2.jpg" /> Şekil 2. Kültür 3 hafta sonra hipokampal nöronların, kırmızı, MAP-2 immün gösteren Temsilcisi görüntü. DAPI boyama, mavi, hücre çekirdeği gösterir. Orijinal büyütme 40x. <img alt="Şekil 3" src="/files/ftp_upload/3965/3965fig3.jpg" /> Nöronal hücre kültürlerinin saflığı gösteren Şekil 3. Western blot. Sıçan ve insan nöronal protein GBM lizatlarının 30 ug elektroforez ile ayrılmış ve standart prosedürler takip 1 Western blot analizi tabi tutuldu. Anti-MAP-2 Hücre Sinyalleşme (Danvers, MA) ile bir tavşan poliklonal olarak, anti-GFAP antikoru Chemicon (Millipore, Billerica, MA) ile bir fare monoklonal, ve fare monoklonal antikoru, anti-Grb2 BD Transduction Laboratories (yapıldı Sparks, MD). Grb2 bir yükleme kontrol olarak kullanılmıştır. </p&gt; <img alt="Şekil 4" src="/files/ftp_upload/3965/3965fig4.jpg" /> Şekil 4. Temsilcisi in vitro 5 gün resimleri (DIV5) sol korteks elde edilen sıçan kortikal nöronların Hazırda E onların diseksiyonu sonrası bir hafta boyunca, 4 ° C + B27. Bir cam çift odacıklı slayt kültüre nöronlar A) Faz kontrast. Orijinal büyütme 20X. B) İmmünofloresan yeşil, nöronal süreçleri MAP-2 ekspresyonu gösteren; kültür astrositik işaretleyici GFAP negatif idi. DAPI boyama, mavi, hücre çekirdeği gösterir. Orijinal büyütme 40x.

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Çıkar çatışması ilan etti.

Rat hipokampal ve kortikal nöronların diseksiyon ve kültür yöntemi burada açıklanan kimyasal olarak tanımlanmış bir orta (Şekil 3) yetiştirilen yaklaşık saf nöronal kültürlerin kullanıldığı deneyler sağlar. Serumsuz medya kültürü neredeyse saf nöronlar için protokolleri daha önce 2,3,4 tanımlanmış olmasına karşın, bizim yöntem yapılan önemli değişiklikler vardır. Geleneksel protokolleri farklı (yani Banker ve ark.) 5, biz TrypLE Express, dah...

<table border="1"><tbody><tr><td> Reaktif </td><td> Konsantrasyon </td></tr><tr><td> Neurobasal </td><td> % 98 </td></tr><tr><td> B27 </td><td> % 2 </td></tr><tr><td> Glutamax </td><td> 0.5 mM </td></tr></tbody></table> Tablo I. Neurobasal/B27 tam medyum. <table border="1"><tbody><tr><td> Reaktif </td><td> Konsantrasyon </td></tr><tr><td> Glikoz </td><td> 16 mM </td></tr><tr><td> Sakaroz </td><td> 22 mM </td></tr><tr><td> HEPES </td><td> 10 mM </td></tr><tr><td> NaCl </td><td> 160 mM </td></tr><tr><td> KCl </td><td> 5 mM </td></tr><tr><td> Na 2 HPO 4 </td><td> 1 mM </td></tr><tr><td> KH 2 PO 2 </td><td> 0,22 mM </td></tr><tr><td> Gentamisin </td><td> 50 mcg / ml </td></tr&gt; <tr><td> Fungizone </td><td> 250 ng / ml </td></tr><tr><td> pH </td><td> 7.4 </td></tr><tr><td> Osmolarite </td><td> 320-330 mOsm </td></tr></tbody></table> Tablo II. Diseksiyon orta. <table border="1"><tbody><tr><td> Reaktif </td><td> Hacim (ul) </td></tr><tr><td> Neurobasal/B27 tam medyum </td><td> 240 </td></tr><tr><td> Tripan Mavi% 0.4 Leke </td><td> 250 </td></tr><tr><td> Toplam </td><td> 490 </td></tr></tbody></table> Tablo III. Çözüm Sayma 50x. <table border="1"><tbody><tr><td> Reaktif </td><td> Şirket </td><td> Cat. numara </td></tr><tr><td> Hazırda D </td><td> Brainbits </td><td> 767171 </td></tr><tr><td> Neurobasal </td><td> Gibco, Invitrogen </td><td> 21103-049 </td></tr><tr><td> B27 </td><td> Gibco, Invitrogen </td><td> 17504-044 </td></tr><tr><td> Fungizone </td><td> Gibco, Invitrogen </td><td> 15290-018 </td></tr><tr><td> Gentamisin sülfat </td><td> Sigma Aldrich </td><td> G1264 </td></tr><tr><td> Glutamax 200 mM </td><td> Gibco, Invitrogen </td><td> 35050 </td></tr><tr><td> TrypLE Express w / o fenol kırmızısı </td><td> Gibco, Invitrogen </td><td> 12604 </td></tr><tr><td> Sitosin-β-D-arabinofuranoside hidroklorür </td><td> Sigma Aldrich </td><td> C6645 </td></tr><tr><td> Poli-D-Lizin </td><td> Sigma Aldrich </td><td> P6407 </td></tr><tr><td> Laminin 1 mg / ml </td><td> Millipore </td><td> CC095 </td></tr><tr><td> HEPES </td><td> Sigma Aldrich </td><td> H3375 </td></tr><tr><td> Tripan Mavi% 0.4 Leke </td><td> Gibco, Invitrogen </td><td> 15250 </td></tr></tbody></table&gt; Tablo IV. Özel reaktifler. <table border="1"><tbody><tr><td> Ekipman </td><td> Şirket </td><td> Cat. numara </td></tr><tr><td> Stereo Mikroskop </td><td> Olympus </td><td> SZ61 </td></tr><tr><td> Büyük Forseps </td><td> FST </td><td> 11022-14 </td></tr><tr><td> İnce uçlu bir pens </td><td> Moria </td><td> MC40B </td></tr><tr><td> Mikro ince uçlu pens </td><td> Moria </td><td> MC31 </td></tr><tr><td> Keskin makaslar </td><td> Roboz </td><td> RS-6820 </td></tr><tr><td> Mikro Kesme makas </td><td> FST </td><td> 91460-11 </td></tr><tr><td> Mikro Eğri makas Dissecting </td><td> FST </td><td> 14067-11 </td></tr><tr><td> Cam 2 odacıklı slaytlar </td><td> Lab-Tek </td><td> 154461 </td></tr><tr><td> 60 mm yemekleri </td&gt; <td> BD Falcon </td><td> 353002 </td></tr><tr><td> 100 mm yemekleri </td><td> Corning </td><td> 430167 </td></tr><tr><td> 15 ml&#39;lik tüplere </td><td> BD Falcon </td><td> 352099 </td></tr><tr><td> 1.5 ml cryo tüp flakon </td><td> Nunc </td><td> 375353 </td></tr></tbody></table> Tablo V. Spesifik ekipmanlar.

isolation and culture of rat embryonic neural cells: a quick protocol

Biz E15-17 rat embriyoları hipokampal veya kortikal nöronların ayırmak ve kültür için hızlı bir yöntem tarif edilir. Prosedürü fare ve insan birincil nöronlar ve sinir progenitörlerin izolasyonu başarıyla uygulanabilir. Ayrışmış nöronlar ila birkaç hafta serum içermeyen aracı madde içinde muhafaza edilir. Bu kültürler nucleofection, immünsitokimya, nükleik asit hazırlanması yanı sıra elektrofizyoloji için de kullanılabilir. Büyük nöronal kültürler da iyi bir verim lentiviral transdüksiyonu ile oranı ve daha az etkin bir şekilde, kalsiyum fosfat ya da bu gibi lipid lipofektamin bazlı yöntem ile transfekte edilebilir.

Watch this Scientific Journal Video about Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol at JoVE.com

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

Biz kemirgen embriyolardan hipokampal ve kortikal nöronlar yalıtmak ve kültürüne hızlı bir metodoloji tanıtılmıştır. Bu protokol bize neredeyse saf nöronal kültürlerin gerekli olduğu deneyler yapmasına imkan tanıyor.

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-and-culture-of-rat-embryonic-neural-cells-a-quick-protocol

Research

JoVE Journal

Neuroscience

38.1K Görüntüleme.  LSU Health Sciences Center - New Orleans. Biz kemirgen embriyolardan hipokampal ve kortikal nöronlar yalıtmak ve kültürüne hızlı bir metodoloji tanıtılmıştır. Bu protokol bize neredeyse saf nöronal kültürlerin gerekli olduğu deneyler yapmasına imkan tanıyor.

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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Embriyonik Omurilik Commissural Nöronlar Diseksiyon ve Kültür

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Nörobilim

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

Neurosphere Testi Yetişkin Fare Nöral Kök Hücre İzolasyonu ve Genişleme

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

Neurosphere Testi kullanarak Embriyonik Fare Nöral Kök Hücre Kültürü oluşturulması

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

İnsan Saç köklerinin arasından Sinir Crest Kök Hücre İzolasyonu ve Kültür

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

Fare Embriyonik motor nöronlar lektin tabanlı İzolasyon ve Kültür

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

İnsan Mantar şeklinde Lezzet papilla hücrelerinin izolasyonu ve Kültürü

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Embriyonik Mürin Nöral Tüp çıkan sinirsel Crest Hücre İzolasyonu ve Kültür

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

Diseksiyon, Kültür ve Analizi<em&gt; Xenopus laevis</em&gt; Embriyonik Retina Doku

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Embriyonik ön beyin gelen Endotel Hücreleri İzolasyon ve Kültürü

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

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 ...