Biz görüntü işleme ve analiz için tavsiye Denis Flipo teşekkür ve David W. Silversides kimin laboratuvar olarak Gata4p [5kb]-GFP fare hattı oluşturuldu. Pilon Araştırma laboratuvarında CIHR, NSERC, FRQS ve FRQNT tarafından finanse edilmektedir.

Etik beyanı Fareleri içeren deneyleri tıbbi araştırmalarda kullanılan hayvanların bakım ve manipülasyon için Hayvan Bakım kurallar Kanada Konseyi takip yapıldı. Hayvanların manipülasyon içeren protokolleri Montreal Quebec Üniversitesi&#39;nden kurumsal etik komitesi tarafından onaylandı (Comite Institutionnel de Koruma des Animaux; referans numarası 0512-R3-650-0513). 1.. Kollajen Jeller hazırlanması Bir doku kültürü kaputu altında, steril bir şekilde çalışır. <ol><li> Standart antibiyotik dahil tam 5x DMEM&#39;yi (Eagle modifiye temel orta) hazırlayın. 20 ml su içinde DMEM tozu ve NaHCO 3 0,925 g 3.37 g çözülür. 0.22 um&#39;lik bir filtreden geçirilerek sterilize edin. Steril 100x penisilin / streptomisin ve steril ısıyla etkisiz hale getirilen fetal sığır serumu, 25 ml 2.5 ml ekle. 4 ° C&#39;de saklayın </li><li> Buz, 800 ul mix, tam 5x 600 ul DMEM ve 1 N NaOH 17 ul Kollajen I çözeltisi (0.02 N, asetik asit, filtre ile sterilize edilmiş olarak 3,77 mg / ml). 3 ml&#39;lik bir son hacme kadar steril su ile seyreltilir. Karışımı ilgili büyüme faktörlerini içerir. Genellikle bağırsak hareketini uyarıcı etki gösterir NCC için 10 ng / ml &#39;de GDNF kullanın. </li><li> , 24 oyuklu bir plaka içerisinde tek bir sıra her çukuruna Deposit yaklaşık 480 ul. Kabarcıkları kaçının. Geri kalan satırlar hücre göçü ile ilgili diğer büyüme faktörlerinin etkisini test etmek için de kullanılabilir. </li><li> Kollajen 37 ° C&#39;de steril bir kuluçka makinesi içinde en az 1 saat süreyle polimerize olsun </li></ol> 2. Hayvanlar 16 diseksiyonu <ol><li> Çiftleşmesi kurmak ve ertesi sabah vajinal fişleri kontrol edin. Gün E0.5, vajinal fiş bulunan gününde öğlen olmak kadın yalıtmak ve E12.5 kadar 12 gün bekleyin. </li><li> Izofluran ile hamile fareler anestezi ve CO 2 inhalasyon yoluyla euthanize. </li><li> % 70 etanol ile fare püskürtün. Karın s kaldırınkin ve diseksiyon makas ile karın boşluğu açmak. </li><li> Buz soğukluğunda PBS (fosfat-tamponlu tuzlu su) ile doldurulmuş, cam bir Petri kabı içine rahim çıkarın. Her bir embriyo implantasyonu sitesi izole etmek için tek tek enine biçimde deciduum şişlikler arasındaki rahim kesin. </li><li> Buz gibi soğuk PBS ile dolu bir cam petri ayrı ayrı her bir implantasyon sitede çalışın. Bir mikroskop altında, rahim kas tabakası kaldırmak için ince forseps kullanır. </li><li> Embriyo ortaya çıkarmak için visseral sarısı kesesi ve amniyon açın. Onlar gelişen bağırsak ile iç içe olarak, plasenta / visseral yumurta sarısının için embriyo katılarak kan damarlarını severing dikkatli olun. </li><li> Boyunda embriyonun başını Sever. </li><li> Sadece koyu kırmızı renkli karaciğer üzerinde, bir embriyonun karın boşluğunda kapalı forseps yerleştirin ve karın boşluğunda bir çapraz açılış yapmak için (basınç uygulayarak durdurmak) kendisi forseps açalım. Doğru açılan forseps aşağı çekintamamen karın açmak için embriyonun arka ucu. </li><li> Karaciğer arkasında bağ dokusu tut ve (kolon anüs bağlı) bağırsakları kırmak için dikkatli olmak, karın dışarı cesareti çekin. </li><li> Embriyonun geri kalanından bağırsaklar serbest bırakmak için kolon kesin. Kesilen kolon boyunca herhangi bir yerde yapılabilir. Sonrası için kuyruk kısmını rezerv. </li><li> Çekum bağ dokusu, bağırsakların daha sonra kalanını didiklemek. Bunu yaparken bağırsakları yara için dikkatli olun. </li><li> Bazı mevcut ise karaciğer ve mide (ince bağırsağın rostral ucunda), hem de mesonephros ve genital sırtlar kesip. </li><li> Ince bağırsak izole. Yine, bağırsak nip için dikkatli olun. Şu andan itibaren, bağırsak dokusunun rosrrocaudal oryantasyon rostral ucunda bulunan keskin eğrilik kullanılarak izlenebilir. EM önce mümkün olduğu kadar kısa bir süre boyunca, oda sıcaklığında PBS içinde küçük bağırsak bırakyatak (adım 3.3). </li><li> Son olarak, doğru embriyonik gelişim evresini belirlemek için her bir embriyo için kuyruk Somitlerin sayısını kaydedin. </li></ol> 3. Embriyonik Bağırsaklar Kesit <ol><li> Embriyo diseksiyonlar ile devam etmeden önce, 100 ml PBS (fosfat-tamponlu tuzlu su) içinde 1.5 g agarozun eritilmesi ve 50 ° C&#39;de tutmak </li><li> Akma bir gömme kalıba eritilmiş agaroz (örneğin, yaklaşık 1/4 boru duvarının çıkarılması için uzunlamasına kesilmiş olan 2 ml mikrosantrifüj tüpü kapalı). Agaroz yaklaşık 42-45 ° C&#39;ye kadar soğumasını bekleyin, o dokunmak için sadece biraz ılık olmalıdır. </li><li> Sadece agaroz katılaşır önce embriyonik bağırsak Embed (bu forseps ipuçları ile değerlendirilebilir ve çevresinde 36-38 ° C meydana gelecektir). Katlanmış rostral sonunda forseps ile bağırsak tutarak, kalıbın uzunluğu boyunca agaroz boyunca çok yavaş çekin. Bu, düz agaroz setleri ise gut tutmaya yardımcı olur. T bırakınkısa sürede taşınıyor karşı başlar yayınlayacak. Bağırsak rostral-kaudal yönde takip edin. </li><li> Agaroz tamamen belirledi sağlamak için, buzdolabı 2-3 dakika içinde kalıp koyun. </li><li> (Açılan Eppendorf tüp dışarı kaydırarak) kalıptan agaroz dışarı atın. Bir bıçak ile, bağırsak dik kesim yapma, her iki uçta aşırı agaroz çıkar. </li><li> Titreşen bir bıçak ile bir mikrotom metal aşamasında aşağı bloke agaroz bağırsak / rostral ucunu Tutkal. Bağırsak / agaroz blok tarafında aşırı agaroz Trim. </li><li> Titreşimli mikrotom odasının metal sahne monte edin. Gerekirse, bu nedenle bağırsak (bu nedenle bıçak dik) mümkün olduğu kadar dik olan sahne açısını ayarlamak. Buz soğukluğunda PBS ile örnek örtün. Tampon yüzeyine altında birkaç milimetre aşağı mikrotom bıçak getir. </li><li> Bu e sağlanması, kuyruk-en ince bağırsağın 200 mikron vibratome enine kesim yapmakach agaroz dilim tam bağırsak bölüm içerir. </li></ol> 4. Bağırsak eksplant kültürü <ol><li> Yavaşça de her ortalarına doğru bir dilim yerleştirerek, forseps ile kollajen jeller üzerine taze kesilmiş bağırsak / agaroz dilimleri düz yatırmak. </li><li> Eksplant üzerinden KKK göçünü sağlamak için, nemli bir% 5 CO2 atmosferinde, 37 ° C&#39;de 3 gün inkübe edilir. </li><li> Forseps ile çok nazikçe kollajen jel kapalı bağırsak / agaroz dilimleri alın. Aşağıdaki jel zarar vermemeye özen gösterin. </li><li> Hücre göçü, model rahatsız emin olmak amacıyla daha sonra inkübasyon ve yıkama adımları sırasında doğrudan kolajen jel dokunmaktan kaçının. Oda sıcaklığında da 1 saat başına (PBS)% 4 PFA (paraformaldehid) 500 ul ile düzeltildi. </li><li> Çukur başına 500 ul DAPI (4 &#39;,6-diamidino-2-fenilindol) çözeltisi (PBS içerisinde 5 ug / ml) ile fiksatif yerine ve oda sıcaklığında 10 dakika inkübe edilir. </li><li> Iyi 3x her yıkayın5 dakika boyunca 500 ul PBS ile. </li><li> Her bir içindeki kolajen jel içine gömülü floresan hücreleri (GFP ve DAPI kanalı) Fotoğraf. </li></ol> 5. Görüntü Analizi Biz eksplant kültürü sonra oluşturulan görüntüleri işlemek ve ölçmek için ImageJ 17 geniş kullanımı yaptı. <ol><li> Floresan hücre fotoğraf gibi aynı büyütmede bir mikrometre slayt görüntüleme başlayın. (Düz Çizgi aracını kullanarak) piksel bir mikron uzunluğunu ölçün. </li><li> (/ Set Ölçeği Analiz; piksel / mikron giriş numarası) ölçeği ayarlayın. </li><li> Her GFP floresan fotoğraf için, 8-bit gri tonlama (Image/Type/8-bit) için görüntü formatını değiştirin. </li><li> Sinyal (Görüntü / Ayarı / Parlaklık / Kontrast) yoğunluğunu ayarlayın. </li><li> Gerekli (Process / Arka Plan çıkarma; yuvarlanan topu yarıçapını ayarlamak) ise arka plan gürültü çıkarın. </li><li> Hücreleri ve hücre kümeleri (Görüntü / / Eşik ayarlama), vurgulamak için bir eşik ayarlayınn kümeleri (Process / Binary / Havza) bölmek için bir dönüm geçerlidir. </li><li> Ilgi (ROI) bölgeleri belirlemek ve hücre sayısı istatistikleri oluşturmak için parçacıklar Analiz (Parçacıklar Analiz / Analiz; set boyut minimum kalan piksel hariç). </li><li> Feret çapı (/ Set Ölçümleri Analiz) dahil ölçüm seçenekleri ayarlayın. </li><li> Grup ROI&#39;ler ve Feret çapını belirlemek için bir bütün olarak ölçmek, hücre yayılması bir göstergesidir (/ Araçlar / ROI Manager / Diğer / VEYA, ardından ROI Müdürü Ekle / ve ROI Manager / Tedbir Analiz). </li></ol>

<ol>
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Yazarlar ifşa hiçbir şey yok.

Biz ex vivo eksplant kültürü tekniği tam GDNF mevcudiyetinde enterik NCC göç potansiyeli ölçmek için nasıl kullanılabileceğini göstermektedir. Bu tür kesin miktar büyük ölçüde daha önce tarif edildiği gibi 11,12,15, 200 mikron kalınlığında bir vibratome bağırsak bölümleri kullanılarak yerine yaklaşık boyutu büyük parça tarafından kolaylaştırılır. Gerçekten de, bu bizi son derece tekrarlanabilir bir ortamda hücrelerin makul bir sayı ile çalışmak için izin...

Nöral tepe hücreleri (NCC) embriyo gelişim boyunca bir çok omurgalı türevler meydana özgü bir geçici hücre türüdür. Bu hücre popülasyonu nöral olmayan ektoderm 1 bitişik sinirsel levha, sınırında ortaya çıkar. Nörülasyon sırasında oluşturan nöral tüpün dorsal kenarı boyunca nöral plak yerlerden KKK arasında eğilme. KKK sonra ayrılması ve uzak nöral tüp göç, epitel-mezenkimal geçiş tabi. KKK onlar tüm enterik sinir sistemi (ENS) oluşturmak sindirim sistemi, bağırsak duvarında gömülü nöral ganglionlarda birbirine bağlı bir ağ dahil olmak üzere çeşitli embriyonik yapılar, kolonize. Geçenlerde 2,3 gözden gibi, birçok genin bu karmaşık yapının geliştirilmesi dahil edilmiştir. ENS çoğu (yani müstakbel Beyin / omurilik sınırındaki civarında) vagal nöral tüp KKK menşeli küçük bir havuz türetilmiştir 4.Bunlar sinir atalarıdır farelerde embriyonik gün (E) 9.0 etrafında foregut ulaşmak ve yaklaşık e15.0 tüm embriyonik bağırsakları kolonize kadar sonra bağırsak mezenşimin içinde caudally göç. Kolon sinir progenitörlerinin Küçük bir alt kümesi, aynı zamanda yukarı çekum 4 ters yönde arka bağırsak işgal sakral NCC, tarafından sağlanır. Vagal ve sakral Hem NCC gerektiren birden fazla göç-çoğalma-, ENS tam oluşmasını sağlamak için hayatta kalma ve farklılaşma-teşvik ipuçları. Bu bağlamda, hayvan modelleri, - özellikle de genetik olarak modifiye edilmiş farenin - birkaç temel hücre dışı ligandların belirlenmesi vesile olmuştur: GDNF (gliyal hücre-türevi nörotropik faktör), endotelin-3, nörotrofin-3, BMP (kemik morfojenik proteinler), netrin , hem de sonik ve Hint Hedgehog (Shh ve Ihh) 5-10. Bunlardan, (transfeksiyon sırasında Rearranged) transmembran reseptör tirozin kinaz yoluyla sinyal RET GDNF inci olarak kabul edilmektedirembriyonik gut ve içinde cazibe ve KKK rehberlik e en kritik yolu. GDNF bağırsak mezenşimin tarafından salgılanan ve RET 11,12 ifade enterik KKK, doğrudan kemoatraktif bir spatiotemporally kontrollü rosrrocaudal degrade oluşturur. Diğer fonksiyonlar arasında, ENS bağırsak duvarında düz kas ile etkileşim yoluyla sindirim sistemi içinde hareketini düzenler. Hirschsprung hastalığında bağırsak sonuçların terminal bölgesinde nöral ganglia olmaması: etkilenen segmentin tonik kasılma tıkanması, sindirilmiş madde birikimi ve üst bağırsak ve karın büyük bir şişkinlik yol açar. Hirschsprung hastalığı yaklaşık 5,000 canlı doğumda oluşur. Enterik KKK rostro-kaudal göç desen Hirschsprung hastalığının etiyolojisinde ana bir faktör olduğuna inanılmaktadır. KKK ve b son bölümünü göç kaynağından kolon, uzakkolonize edilmesi Owel, ENS oluşumunda hatalarına karşı en hassas olduğunu. Enterik NCC göç onun önemli rol ile uygun olarak, GDNF / RET sinyal bozulması Hirschsprung hastalığı 13&#39;ün ana bilinen genetik bir nedenidir. [5kb]-GFP 14 Gata4p adında - - Göçmen KKK Yeşil Floresan Protein (GFP) ile etiketli olduğu daha iyi KKK ve ENS gelişimini incelemek için, biz bir transgenik fare hattı oluşturulur. Gelecek artık, GDNF gibi çeşitli büyüme faktörlerinin varlığında enterik NCC göç potansiyeli hassas miktar tayinine izin veren diğer gruplara 11,12,15 tarafından yayınlanmış çalışmadan adapte edilen bir ex vivo hücre göçü deneyi, mükemmel.

<table border="1">
	<tbody>
		<tr>
			<td>DMEM powder</td>
			<td>Wisent</td>
			<td>219-010-XK</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Bioshop</td>
			<td>SOB999</td>
			<td>Biotechnology grade</td>
		</tr>
		<tr>
			<td>Steriflip vacuum filtration system (0.22 micron)</td>
			<td>EMD Millipore</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin-Streptomycin solution, 100x</td>
			<td>Wisent</td>
			<td>450-201-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Wisent</td>
			<td>095-150</td>
			<td>High quality grade</td>
		</tr>
		<tr>
			<td>Collagen I</td>
			<td>BD biosciences</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Bioshop</td>
			<td>SHY700</td>
			<td>Diluted from 10 N stock then sterile-filtered</td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>Cedarlane</td>
			<td>CLCYT305</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 24-well Plate</td>
			<td>BD biosciences</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td>Fisher Scientific</td>
			<td>089515</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Petri dish</td>
			<td>VWR</td>
			<td>89000-306</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma</td>
			<td>P5493</td>
			<td>Cell culture grade</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Leica</td>
			<td>M125</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Bioshop</td>
			<td>AGA001</td>
			<td>Biotechnology grade</td>
		</tr>
		<tr>
			<td>Surgical blade</td>
			<td>Feather</td>
			<td>21</td>
			<td></td>
		</tr>
		<tr>
			<td>All Purpose Instant Krazy Glue Pen</td>
			<td>Krazy Glue</td>
			<td>KG824</td>
			<td></td>
		</tr>
		<tr>
			<td>HM 650V Vibrating-Blade Microtome</td>
			<td>Thermo Scientific</td>
			<td>920110</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9564</td>
			<td></td>
		</tr>
	</tbody>
</table>

a quantitative cell migration assay for murine enteric neural progenitors

Nöral krest hücreleri (KKK) dorsal nöral tüp kaynaklanan ve gelişmekte olan omurgalı embriyo boyunca yoğun göç geçici ve multipotent hücre popülasyonu vardır. Çevresel glia ve nöronlar sağlamanın yanı sıra, NCC melanositler gibi kranyo-yüz iskeletinin en üretir. NCC göç ve farklılaşma nöral tüp ve bölgesel olarak farklı hücre dışı uyaranlara maruz kaldıkları boyunca eksenel kökenli bir kombinasyonu ile kontrol edilmektedir. Hücre dışı ligandlar Bu katkı enterik sinir sistemi (ENS), karmaşık bağlantılı (diğer şeyler arasında) lokal olarak kontrol nöral ganglia ağı bağırsak, kas hareketi ve intestinal motilite oluşumu sırasında, özellikle belirgindir. Kaudal moda bir rostral - - müstakbel bağırsak tüm uzunluğu ENS çoğu kolonize etmek için uzun bir yolculuğa üstlenmek KKK başlangıçtaki küçük bir havuz türetilmiştir. Bilinen çeşitli sinyal yolları arasında,enterik KKK kolonizasyonu etkileyen, GDNF / RET sinyal en önemli olarak kabul edilmektedir. Nitekim, bağırsak mezenşimin tarafından RET ligand GDNF&#39;nin spatiotemporally kontrollü salgılanması embriyonik gut ve içinde cazibe ve RET-ifade enterik KKK rehberlik için başlıca sorumludur. Burada, GDNF gibi çeşitli büyüme faktörlerinin varlığında enterik NCC göç potansiyeli hassas miktar tayinine izin veren, floresanla işaretlenmiş NCC, sahip olan bir transgenik fare hattı yararlanarak, bir ex vivo hücre göçü deneyi tarif eder.

Aşağıdaki sonuçlar (Şekil 1) burada tarif edilen teknik ile elde edilebilir ne temsil etmektedir. Büyüme faktörleri (örneğin GDNF) kullanılması GFP-ifade eden intestinal eksplant dışarı ve kolajen jel içine enterik NCC (Şekil 2) göçünü teşvik eder. Bazı hücreler, büyüme faktörlerinin yokluğunda eksplant çıkması rağmen, bu çoğunlukla GFP-etiketli değildir ve pasif girişi temsil eder. Bu doku hala yoğun floresan hücreler tarafından doldurulu...

Watch this Scientific Journal Video about A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors at JoVE.com

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Çeşitli büyüme faktörlerinin varlığında enterik nöral tepe hücre göçü potansiyelinin hassas miktar tayinine izin veren bir ex vivo hücre göçü deneyi mevcut.

Kemirgen Enterik Sinir progenitörlerin için Kantitatif Hücre Göç Tahlili

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

a-quantitative-cell-migration-assay-for-murine-enteric-neural

Research

JoVE Journal

Neuroscience

9.2K Görüntüleme.  UQAM.  Çeşitli büyüme faktörlerinin varlığında enterik nöral tepe hücre göçü potansiyelinin hassas miktar tayinine izin veren bir ex vivo hücre göçü deneyi mevcut. 

Video: A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

Kronik İki foton için İnce kafatası Pencere Tekniği In vivo</emNöro-inflamasyon Modelleri murin mikroglia> Görüntüleme

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

Nörobilim

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 ...

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

Hücre Testi Şekillendirme Sinir-Koloni: Sinir Progenitör Hücreler Bona Fide Sinir Kök Hücreler Ayırım Bir Testi

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

Bir Modifiye Zigmond Odası Testi kullanılarak Trunk Sinir Crest Hücre Göç Analizi

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Tüm Dağı Muscle Analizi ile tekrarlanabilir Fare Siyatik Sinir Ezilme ve Rejenerasyon daha sonraki Değerlendirme

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

Çapraz türler Nakli Sinir Crest Göç ve Farklılaşma Analiz

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Neuroblast Test: Akım Sitometri Kullanımı Farklılaşan Nöral Kök Hücre Döl nöronal Progenitör Hücrelerinin üretilmesi ve zenginleştirilmesi için bir Testi

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

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 ...

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

Dıştan Uygulanan Doğru Akım Elektrik Alanında Nöral Prekürsör Hücre Göç Kinetik Analizi için galvanotaksis Assay

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.