Yazarlar yardım flow sitometri James Oesterling teşekkür etmek istiyorum. Bu araştırma ABD Ulusal Kanser Enstitüsü (R01-CA178380, ZY) ve ABD Ulusal Sağlık Enstitüleri Doktora Sonrası eğitim bursu (5T32CA009035-37, LWY) bir hibe ile desteklenmiştir.

Bu protokolde hayvanların kullanımı Fox Chase Kanser Merkezi&#39;nde Hayvan Bakım ve Kullanım Kurulu tarafından onaylanan prosedürlere uygun olarak gerçekleştirilmiştir. Göstergeler, Çözümler ve lameller 1. Hazırlık <ol><li> # 5 ince forseps temiz iki takım, 7. bir set ince kavisli forseps, büyük bir cerrahi makas, bir Microdissecting makas, bir spatula ve bir delikli kaşık. Kendi kendine yapışan bir sterilizasyon kese ve otoklav içindeki aletleri yerleştirin. Kullanıma hazır olana kadar kese aletleri tutun. </li><li> % 3 düşük erime sıcaklığına sahip bir agaroz çözelti hazırlayın. <ol><li> Kabarcıklar ortaya çıkıncaya kadar steril Dulbecco Fosfat Tamponlu Salin (DPBS) içinde 50 ml 1.5 g agarozun Add mikrodalga ve ısı yerleştirin. Tüm agaroz kadar girdap içinde eritildi ve yeniden ısıtma çözülene. Kümeleri görüldüğünde ısıtmadan önce bir karıştırma çubuğuna sahip olan çözüm karıştırın. </li><li> Bir 37 ° C su banyosunda solüsyon deposu kadar gerekli. </li><li> Uzun süreli Saklama Sistemi İçine ay boyunca oda sıcaklığında ya da 4 ° C &#39;de gelecekte kullanılmak üzere agaroz çözüm saklayın. Sıvılaştırılması için mikrodalga ısıtın. Agaroz çözeltinin tekrar ısıtılması için ılık steril damıtılmış su ile başlangıç ​​ağırlığı korumak için ısıtmadan önce şişeyi tartın. </li></ol></li><li> Daha önce tarif edildiği gibi 2 papain temelli hücre ayrılması için aşağıdaki çözümleri hazırlayın: <ol><li> Steril fenol kırmızı DPBS ihtiva eden papain (100 U / ml), sistein (0.2 mg / ml) ve DNAse (250 U / ml) ile bir papain çözeltisi. </li><li> Büyükbaş hayvan serum albümini ile bir ovomukoid çözeltisi (BSA, 8 mg / ml), soya fasulyesi tripsin önleyici (8 mg / ml)) ve DNaz (250 U / ml) steril kırmızı olmayan fenol ihtiva eden DPBS. Filtre hem çözümler sterilize ve fenol kırmızı içeren DPBS orijinal rengini geri dönmek için pH ayarlamak. </li></ol></li><li> Hücre kültürü ortamı (NB / B27) hazırlanması: 1 mM sodyum piruvat, 2 mM L-glutamin,% 1 penisilin-streptomisin (P / S), B27 eki ile bir bazal ortam ek. FilteR, sterilize ve ışıktan korur. Kullanımdan önce, 37 ° C&#39;de ve ortam% 5 CO2 dengelenmesi. </li><li> FACS tamponu hazırlayın:% 5 fetal sığır serumu, DPBS (FBS). 47.5 ml DPBS 2.5 ml FBS ekleyin. </li><li> Poli-D-lisin (PDL) kaplı lamelleri hazırlayın. <ol><li> Otoklav Yeni cam lamelleri. </li><li> Kaplayın PDL lamelleri (steril damıtılmış su içinde 100 ug / ml) ve 37 ° C&#39;de 2 saat boyunca inkübe edilir ve gece boyunca oda sıcaklığı. </li><li> Yıkama steril damıtılmış su ile lamelleri. NB / B27 medya ile bir kez yıkayın. Lamelleri kullanmak için önce doku kültürü kaputu tamamen kurumasını bekleyin. </li></ol></li></ol> Doku Dilimleri 2. Diseksiyon ve Hazırlık <ol><li> Diseksiyon gününde, emici bir ped ile% 70 etanol ve kapak ile diseksiyon alanı silin. </li><li> Sterilizasyon kesesinden diseksiyon aletleri çıkarın. Buz soğuk, buz üzerinde küçük bir petri ve yere steril DPBS /% 1 P / S ekleyin. </li><li> Decapitate P4 Math1-GFBüyük cerrahi makas ile P / Nestin-OBP fare yavrular sonra ince kavisli bir forseps ile kafa basılı tutun. Cilt kaldırın ve yavaşça ince forseps daha sonra bir spatula kullanarak kafatası beyin kepçe ve DPBS /% 1 P / S içeren Petri kabı aktarmak # 5 ile kafatasını soymaya. <ol><li> # 5 ince forseps kullanarak, dikkatli bir beynin geri kalanından beyincik ayrı. Mümkün olduğunca çabuk bir defada bir yavru incelemek için emin olun. </li><li> Moleküler ve işlevsel analiz için yeterli lif düğümlerinin elde edilmesi için 8 cerebella az toplayın. </li></ol></li><li> % 3 düşük erime sıcaklığına sahip bir agaroz çözeltisiyle 2 x 2 x 2 cm büyüklüğünde bir gömme kalıp doldurun. Agaroz sıcaklığı fazla 37 ° C olduğundan emin olun. </li><li> Laboratuar doku yavaşça dabbing beyincik fazla sıvıyı çıkarın. Dikey konumda kalıba beyincik yerleştirin. Hızlı bir şekilde katılaşmasına izin vermek için buz üzerinde kalıp yerleştirin. Blok başına ~ 4 cerebella kullanın. </li><li> Li edinintitreşimli bıçak vibratom ile Ving doku bölümleri. Bir jilet ile serebellası içeren agaroz blok Trim. Dikey konumda yönlendirilmiş cerebella ile vibratome plakasına agaroz blok tutkal. </li><li> Altında buz-soğuk steril DPBS&#39;de / 1% P / S ve yer buz ile vibratome tepsiyi doldurun. <ol><li> Beyincik tüm uzunluğu boyunca 600 um kesitler halinde kesilmiştir. Delikli bir kaşık kullanarak buz tepsiden bölümleri toplayın. Buz üzerinde DPBS /% 1 P / S ile dolu bir Petri tabağına yerleştirin bölümleri. </li></ol></li></ol> 3. Mikrodisseksiyon ve Hücre Ayrılma <ol><li> Bir floresan mikroskop altında doku dilim içeren Petri kabı yerleştirin. <ol><li> Dikkatle (Şekil 1 &#39;de noktalı çizgi bakınız) CFP + moleküler tabakası ve GFP + EGL arasında kesilmesi yoluyla ince pensler kullanılarak bir serebellar bölümün geri kalanından ayırmak EGL. Neden olacaktır, moleküler tabakanın herhangi bir bölümünü dahil değil dikkatli olunNestin-ifade Bergmann glia kontaminasyon. </li><li> Mümkün olduğunca çabuk bu adımı tamamlayın ve hücre ölümünü önlemek için buz üzerinde doku tutun. </li><li> Bir sonraki aşamaya geçmeden önce dokuyu çevreleyen agaroz çıkarın. </li></ol></li><li> Daha önce tek bir hücre süspansiyonu elde etmek için tarif edildiği gibi 2 papain tabanlı bir protokol kullanarak microdissected EGL doku Digest. <ol><li> FACS üzerinden CFP-pozitif hücrelerin toplanması için FACS tampon hücreleri yeniden askıya. </li></ol></li></ol> 4. Hücre Sıralama ve Kaplama <ol><li> Uygun bir CFP filtresini içeren ve buz üzerinde FACS tamponu hücreleri toplamak sitometresi steril, yüksek hızlı akışı kullanılarak CFP floresan için sıralama hücreleri. Her P4 beyincik yaklaşık 100.000 NEPS edinin. </li><li> 5 dakika boyunca 300 xg&#39;de Santrifüj hücreleri. Daha fazla deney için moleküler analiz veya kültür için hemen hücreleri kullanır. </li><li> Kültür hücreleri, önceden ısıtılmış NB / B27 m hücreleri yeniden askıyaPDL kaplı lamelleri edia ve plaka daha önce 2 nitelendirdi. </li></ol>

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Yazarlar ifşa hiçbir şey yok.

Burada anlatılan yöntem mikrodiseksiyon moleküler ve fonksiyonel analizlerde kullanım için canlı hücreler saf bir popülasyonu izole etmek mümkün ilk işlemdir. Li ve ark. 9 ile gösterildiği gibi, Bu yöntemle elde edilen NEP kültürlenmiştir ve çeşitli deneyler dahil edilir ve olduğu gibi yüksek saflıkta, aynı zamanda mikro-dizi analizi için de kullanılabilir edilebilir. Bu mikrodisseksiyon tekniğiyle ustalaşmanın zaman çok önemlidir....

Beyincik, çok hücreli bir katman, her biri farklı hücre tiplerinde meydana gelir. Moleküler tabaka ve purkinje katman sırasıyla Bergmann glia ve purkinje nöronları içeren ise gelişme sırasında, EGL granül nöron öncüleri (kişi başına milli geliri) çoğalan içerir. Beyincik içinde derin nöral kök hücreler (NSC&#39;lerde) ve oligodendrositleri 1 içeren beyaz madde, yatıyor. Sonic dikenli proteini (Shh) serebellar gelişme düzenlenmesinde önemli bir rol oynadığı ve özellikle de, kendi reseptörü olan yamalı (PTC), Shh, 2-4, sinyal bir negatif düzenleyicisi bağlanarak kişi başına milli geliri çoğalmasını teşvik eder. Sus sinyal anormal aktivasyonunun Medulloblastom (MB), çocuklarda 5,6 en sık görülen kötü huylu beyin tümörü oluşturur. Ptc mutant MB transgenik fare modelleri MB çalışma için güçlü araçlar vardır ve insan MB 7,8 benzeyen tümörler gelişebilir. Kullanarak bufarenin, bu kişi başına milli geliri kirpi tipi MB 7 kökenli hücre olduğu keşfedildi. Ayrıca, ek olarak kişi başına milli geliri, yakın zamanda da MB ortaya çıkmasına neden olabilir gelişen beyincikte de EGL içinde nöronal progenitörlerin benzersiz bir popülasyonu tespit ettik. Bu hücreler, tip VI filaman protein Nestin yüksek seviyelerde ifade etmek ve NEP 9 olarak adlandırılır. NEP EGL derin bölümü içinde yer alan ve sadece yenidoğan serebellar gelişme sırasında geçici olarak mevcut olurlar. Onlar kişi başına milli geliri olarak granül hücre soyu kararlıdır ama onlar sakin ve Math1, konvansiyonel kişi başına milli geliri 10 köklü bir işaretleyici ifade yok gibi farklıdır edilmektedir. Buna ek olarak, NEP onları MB tümörogenez için yeni bir kökeni yapar Şşş sinyal yolunun 9 aktivasyonu sonrası MB yol daha verimli kişi başına milli geliri verir. Biz daha önce mikrodisseksiyon tekniği ile doğum sonrası 4. günde (P4) de serebellar EGL gelen NEPS izoleburada 9 nitelendirdi. Dokunun direkt mikroskopik ile mikrodiseksiyon serebellar EGL belirli diseksiyon sağlar. Nestin da serebellar beyaz cevher ve moleküler tabaka 1,7,11,12 Bergmann glia ile NSC&#39;lerde ifade ve analizleri etkileyebilecek gibi o, bu hücreler dahil edilmemesi çok önemliydi gibi bu gereklidir. Microdissected dokudan izole edilen hücreler moleküler analiz için hemen kullanılabilir ya da daha fazla uygulama için kültürlenebilir. Özellikle EGL Microdissecting yardımcı olmak için ve daha fazla nepler arıtmak için Math1-GFP (yeşil floresan protein) farenin Nestin-CFP (mavi floresan protein) fareler ile çaprazlandı. Math1 spesifik kişi başına milli geliri ve GFP ifadesi ile anlatılmak istenen, transkripsiyon faktörü EGL 9,13 açıkça görülebilir. Nestin-CFP farenin CFP nükleer şeklinde ifade ve kolayca moleküler tabakanın 9,14 olarak görselleştirilebilir. Birlikte, GFP ve CFP ifade EGL mikrodiseksiyonu için sınırlarını oluşturmak (Şekil 1). CFP pozitif NEP daha sonra kesilmiş EGLs enzimatik ayrışma sonrasında, FACS ile izole edilmiştir. Şu anda, EGL hücreleri izole etmek için en çok kullanılan yöntem, tüm beyincik dokusuna 15,16 Percoll gradient santrifüjleme. Ancak bu metot, tam olarak moleküler tabakanın beyaz maddeden Nestin + hücre popülasyonlarının dahil yapamaz ve dolayısıyla neps çalışma için kullanılamaz. Ilgi hücreleri çıkarmak için lazer enerjisinin transferini kullanan lazer yakalama mikrodiseksiyon, heterojen bir doku 17,18 içinde belirli hücreleri izole etmek için kullanılabilir, ancak yakalanan hücreleri sadece DNA, RNA ve proteinin geri kazanılması için kullanılan ve değildir başka bir yöntemdir edebilmek yetiştiriciliğine. Bu protokol, özellikle EGL gelen NEP&#39;lerin bir canlı, saf bir nüfusunu yalıtmak için bir yol sağlar.Bu teknik aynı zamanda tanınan anatomik mimari veya flüoresan etiketli hücre / alanlara sahip farklı doku türlerine uygulanabilir. Bu nedenle, hücre izolasyon protokolleri, bu yeni mikrodiseksiyon tekniği içeren en büyük avantajı, hücreler, doku komşu kirletici ortadan kaldırmak için özel doku bölgeleri izole edilebilir ve hücreler, diğer uygulamalar için veya kültür analizi için hemen elde edilebilmesidir.

<table border="0" cellpadding="0" cellspacing="0" width="572">
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			<td height="42" style="height:42px;width:215px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Neurobasal media</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">21103-049</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:215px;">PDL</td>
			<td style="width:71px;">Millipore</td>
			<td style="width:119px;">A-003-E</td>
			<td></td>
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			<td height="42" style="height:42px;width:215px;">ultra pure low melting temperature agarose</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">16520-050</td>
			<td></td>
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			<td height="21" style="height:21px;width:215px;">DPBS</td>
			<td style="width:71px;">Gibco</td>
			<td align="right" style="width:119px;">14190144</td>
			<td></td>
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isolation of distinct cell populations from the developing cerebellum by microdissection

Mikrodisseksiyon bir doku spesifik bölgelerini izole ve komşu bölgelerde hücresel kaynaklardan kirlenmeyi ortadan kaldırabilir yeni bir tekniktir. Bu yöntem, ilk çalışmada kullanılmıştır Nestin salgılayan progenitorlar (döküntüler), serebellar dış germinal tabakadan (EGL) hücrelerin yeni tespit edilen bir popülasyon. Floresan-aktive edilmiş hücre tasnifi (FACS) ile kombinasyon halinde kullanılması mikrodiseksiyon, neps saf nüfus serebellumda EGL geleneksel granül nöron habercilerden ve diğer kirletici Nestin salgılayan hücrelerin ayrı olarak toplanmıştır. Mikrodiseksiyonu olmadan, NEP&#39;lerin fonksiyonel analizleri gibi Percoll santrifüj ve lazer yakalama mikrodiseksiyon olarak kullanılabilir mevcut yöntemleri ile mümkün olmazdı. Bu teknik aynı zamanda tanınan bölgeleri veya floresan ile etiketlenmiş ya da hücreleri içeren çeşitli dokularda kullanılmak için de uygulanabilir. Bu microdissectio en önemlisi büyük bir avantajdırn, teknik izole edilmiş canlı hücreler ve başka tarif edilen yöntemler ile mümkün değil ayrıca deney için kültürlenebilir olmasıdır.

Şekil 1A&#39;da gösterildiği gibi, serebellar dilim hangi serebellar EGL GFP ifade kişi başına milli geliri hakim ve moleküler tabaka CFP pozitif glial hücreler tarafından zenginleştirilmiş P4 Math1 GFP / Nestin-CFP farelerden hazırlandı. (Şekil 1A kesikli hattı boyunca) EGL derin kısmında bulunan lif düğümlerini izole etmek için, dilimler EGL ve moleküler tabaka arasında microdissected edildi. Disseke EGLs (Şekil 1B) daha sonra...

Watch this Scientific Journal Video about Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection at JoVE.com

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Nestin-salgılayan progenitorlar gelişen beyincikte nöronal progenitörlerinin yeni tespit edilen bir gruptur. Floresan-aktive edilmiş hücre sıralama ile kombinasyon halinde burada sunulan mikrodiseksiyon tekniği kullanarak, bu hücre popülasyonu, diğer beyincik bölgelerinin herhangi bir bulaşma ile saflaştırılabilir ve daha ileri araştırmalar için kültürlenebilir.

Mikrodisseksiyon tarafından Gelişmekte Serebellumunda farklı olarak hücre popülasyonlarının İzolasyonu

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. ...

isolation-distinct-cell-populations-from-developing-cerebellum

Research

JoVE Journal

Neuroscience

9.5K Görüntüleme.  Temple University Health System.  Nestin-salgılayan progenitorlar gelişen beyincikte nöronal progenitörlerinin yeni tespit edilen bir gruptur. Floresan-aktive edilmiş hücre sıralama ile kombinasyon halinde burada sunulan mikrodiseksiyon tekniği kullanarak, bu hücre popülasyonu, diğer beyincik bölgelerinin herhangi bir bulaşma ile saflaştırılabilir ve daha ileri araştırmalar için kültürlenebilir. 

Video: Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

Bozulmamış Retina Elektrofizyolojik Karakterizasyonu GFP-ifade eden hücre popülasyonlarının

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

Nörobilim

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 ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

Travmatik Beyin Hasarı Sonrası Nöronlar veya gen ekspresyon analizi için Tek Nöron zenginleştirilmiştir Populasyonlarının Lazer Yakalama Mikrodisseksiyon

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

Lazer Yakalama Mikroseksiyonu ile Fare Omurilik Motor Nöron Hücre Gövdelerinden Transkriptomik Analiz için RNA Üretimi

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Gelişmekte Fare Embriyonik Cortex mitoz Canlı Görüntüleme

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

Lazer Yakalama Mikrodisseksiyon - Bireysel Dopamin Nöronlar İzolasyonu ve Tüm ventral tegmental Area bir Gösteri

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

Tek hücreli Monitor hücre biyolojisi ve birden çok sinir nüfus Lineage ilerlemesini izleme ve ardından görüntüleme yaşamak

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

Lazer-yakalama mikrodiseksiyon optimizasyonu gelen insan dokusu taze donmuş enterik gangliyon yalıtım için

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or regions from heterogenous tissues. Captured product can be used in a variety of molecular methods for protein, DNA or RNA isolation. However, preservation of RNA from postmortem human brain tissue is especially challenging. Standard visualization techniques for LCM require histologic or immunohistochemical staining procedures that can further degrade RNA. Therefore, we designed a stainless protocol for visualization in LCM with the intended purpose of preserving RNA integrity in post-mortem human brain tissue. The Purkinje cell of the cerebellum is a good candidate for stainless visualization, due to its size and characteristic location. The cerebellar cortex has distinct layers that differ in cell density, making them a good archetype to identify under high magnification microscopy. Purkinje cells are large neurons situated between the granule cell layer, which is a densely cellular network of small neurons, and the molecular layer, which is sparse in cell bodies. Because of this architecture, the use of stainless visualization is feasible. Other organ or cell systems that mimic this phenotype would also be suitable. The stainless protocol is designed to fix fresh-frozen tissue with ethanol and remove lipids with xylene for improved morphological visualization under high magnification light microscopy. This protocol does not account for other fixation methods and is specifically designed for fresh-frozen tissue samples captured using an ultraviolet (UV)-LCM system. Here, we present a full protocol for sectioning and fixing fresh frozen post-mortem human cerebellar tissue and purification of RNA from Purkinje cells isolated by UV-LCM, while preserving RNA quality for subsequent RNA-sequencing. In our hands, this protocol produces exceptional levels of cellular visualization without the need for staining reagents and yields RNA with high RNA integrity numbers (&#8805;8) as needed for transcriptional profiling experiments.

This protocol uses a stain-free approach to visualize and isolate Purkinje cells in fresh-frozen tissue from human post-mortem cerebellum via laser capture microdissection. The purpose of this protocol is to generate sufficient amounts of high-quality RNA for RNA-sequencing.

Yüksek kaliteli lazer RNA izolasyonu için paslanmaz bir protokol yakalama insan öldükten sonra beyincik Microdissected Purkinje hücreleri

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or ...

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 ...