NIH hibe HD-32.116, Sandler Aile Vakfı, John Bowes Kök Hücre Fonu, MEXT, MHLW ve HFSP Yardımcı tarafından desteklenen çalışın. ZM Carlos Baldoceda Vakfı ve UCSF Krevans Bursu tarafından desteklenmektedir. AA-B. Heather ve Melanie donatılmış Başkanı Muss tutar.

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Ventrikül ve subventricular bölgelerde nöron çalışmaların çoğu bu bölgelerde mikroanatomisinin ve hücresel ilişkileri incelemek için klasik kesit alma teknikleri dayanıyordu. Burada sonra anti-mitotik tedavi 6 ve en son kesin apikal çalışma için kullanılan aşağıdaki SVZ progenitör nüfusun yenilenme incelemek için kullanılan ilk SVZ 1 neuroblasts göçmen zincirleri ağ analiz etmek için kullanılan alternatif bir yöntemdir, tarif ve yetişkin SVZ sinir kök hücreleri 2,3,4 bazal hücre-hücre etkileşimleri. İlginçtir ki, bu tekniğin yetişkin SVZ sinir kök hücreleri, ya da tip B1 hücreleri, farklılaştırılmış olmayan bölünmesi ependimal hücreler ile karışık bir neuroepithelium parçası olduğunu ortaya koymuştur. En yüze görüntüleme wholemounts kullanarak bu karışık neuroepithelium ependimal hücreler 4 büyük apikal yüzeyleri ile çevrili tip B1 hücrelerinin apikal sonlar oluşan fırıldak mimarisi olduğunu göstermiştir. Bu tr-yüz analizi hücreleri vasküler bir niş iletişime ventrikül yüzeyinde apikal sonlar ve bazal süreçleri ile oluşan embriyonik ve yetişkin beyinlerinde sinir kök hücrelerinin köken anlayışımıza açıklık vardır. Bu bulgular klasik kesit alma teknikleri kullanarak, neredeyse imkansız olurdu. Wholemounts da ventrikül temas apikal süreci ile nöral kök hücrelerin belirlenmesini kolaylaştıracaktır. Bu kök hücreler için daha spesifik belirteçler olarak, wholemounts sinir kök hücre davranışının belirlenmesi ve analiz ayrılmaz bir parçası olacaktır. Lateral ventrikül duvarları Wholemounts ependimal hücreler düzlemsel polarite çalışmak için ideal bir perspektif sağlamaktadır. Ependimal hücreler koordineli bir şekilde BOS itmek işlevi ventriküller astar multiciliated hücrelerdir. Wholemount tekniği ile, tüm ependim epiteli en-face maruz kalmaktadır ve posterior ve dorsal ventral sınırları boyanmış ve onun anterior kapsamlı incelenebilir. Ayrıca, ependim akış testleri akut disseke, canlı wholemounts sağlam ependim kirpikler tarafından oluşturulan düzlemsel polarize akış göstermektedir. Son çalışmalar wholemount yaklaşımları kullanarak bu ependim düzlemsel polarite 5 hücresel belirleyicileri ortaya çıkardı. İlginçtir ki, wholemount çalışmalar da ependim oluşturulan BOS akım SVZ 7 genç nöronların göç kılavuzu chemorepellents gradyanlar kurar düşündürmektedir. Bu nedenle başlangıçta göçmen nöron zincirleri ağ tespit Wholemount yaklaşımlar zinciri göç düzenleyen mekanizmalar açılımlar sağlamaya devam etmektedir. Wholemount görüntüleme VZ ve SVZ Analizi, hem de gelecekteki çalışmaları ve mevcut çalışmaları anlayışımız açıklığa kavuşturmak için bir yol için yeni bir yaklaşım ekliyor. Örneğin, yeni bir çalışmada, yetişkin SVZ nöral kök hücreler ventrikül 8 ile temas CD133 + / CD24-hücreler olduğunu düşündürmektedir . Bölümlerde kendi immün dayanarak, bu yazarların bu hücrelerin bir ICSI&#39;nin multiciliated ependimal hücreler olduğunu iddia etti. Ancak, bizim çalışmamızda tüm ependim epitel daha kapsamlı bir görünüm verir wholemount yaklaşım kullanarak, bulduğu tüm ependimal hücreler ifade CD24 ve tek ventrikül temas B1 tipi bir alt kümesi + / CD24-CD133 hücrelerin hücreleri 4. Ayrıca, wholemount teknikle, yetişkin beyin 9 sinir kök hücrelerinin son açıklanan mozaik organizasyon inceleyerek gelecekteki çalışmalarda faydalı olmayı vaat ediyor . Çeşitli çalışmalar, yetişkin bir insanın beyninin nöral kök hücreler homojen bir nüfus olduğunu göstermiştir, fakat bölgesel belirtilen ve normal koku alma ampul internöronlar yalnızca belirli alt tipleri üretmek. Bu çalışmalar, nöral kök hücrelerin farklı alt popülasyonlar özel transkripsiyon faktörleri 10,11,12,13,14 ve / veya dorsal-ventral ve ön-arka kapsamlarını boyunca bölgesel localisation ifade ayırt edilebilir olduğunu önerdi lateral duvar 9,15,16. Yetişkin nöral kök hücrelerin bölgesel belirtilen alt popülasyonlar daha moleküler belirteçlerin tespit olarak, wholemount görüntüleme ventrikül duvar boyunca bu farklı progenitör etki bölümlenmesi kapsamlı bir bakış vermelidir. Burada sunulan wholemount diseksiyon ve görüntüleme teknikleri de ventriküler duvarları embriyo analiz etmek için kullanılıyor olabilir. Embriyonik lateral duvar diseksiyonu, adım-adım, aynı şekilde yapılır. Zorluk seviyesinde sadece ufak farklar vardır; embriyonik ventriküller diseksiyonu kolaylaştırır görece daha büyük, ancak doku manipülasyonu daha zor hale yumuşaktır. Özellikle lateral ventrikül benzer bir poz Embry kullanılabiliros kortikal kortikal nöron çalışmada ventrikül duvar incelemek. Son kanıtlar, bu asimetrik centrosome miras kortikal nöron 17 sırasında ventrikül yüzeyinde radyal glia korur göstermektedir . En yüze radyal glial apikal yüzeyleri görüntüleme bu bölünen hücreleri içinde centrosomes asimetrik miras nasıl içgörüler sağlayabilir. Özellikle en hassas becerisi içeren bu teknikleri, olduğu gibi, ustalık pratik gerektirir. Ancak, daha iyi sonuçlar için anahtar diseksiyonu birkaç elemanları vardır: 1) aydınlatma gölgeler oluşturmak için örnek aydınlatma ayarlama gibi forseps kullanarak), aksi takdirde 2 nispeten homojen doku diseksiyonu sırasında paha biçilmez bir kontrast sağlar birlikte çimdik veya doku almak için kullanılan bu tekniğin iki böcek pimleri forseps asla, ama manevra pimleri dokuyu stabilize etmek için sürekli yeniden olabilir), nazik retraksiyon bir denge 3 kesme ve kesme bıçak olmamalıdır olarak kullanılır sadece kesmek için değil, aynı zamanda bu diseksiyon çoğunluğu aslında sadece aralıklı kesme nazik retraksiyonu ile gerçekleştirilir olduğunu hatırlayarak, medial ve lateral duvarlar ayrı nazik retraksiyon sağlamak için kullanılır.

the subventricular zone en-face: wholemount staining and ependymal flow

Lateral ventriküllerin duvarları en büyük yetişkin memelilerin beyin germinal bölge içerir. Bu duvarlar subventricular bölgesi (SVZ), davranış ve sinir kök hücrelerinin, yetişkin nöron düzenlenmesi anlamak için yoğun çalışılan bir model sistem. Geleneksel olarak, bu çalışmalar, histolojik analizi için klasik kesit teknikleri konusunda yararlanmıştır. Burada, bu germinal bölgenin kapsamlı, en-yüz görünümü sağlar wholemount tekniği, alternatif bir yaklaşım sunuyoruz. Bölümlerini, wholemounts SVZ içinde, tam hücre mimarisini ve hücresel ilişkileri korumak. Bu yaklaşım son zamanlarda yetişkin sinir kök hücreleri, ya da tip B1 hücreleri, lateral ventriküller astar farklılaştırılmış ependim hücreleri ile karışık bir neuroepithelium parçası olduğunu ortaya koymuştur. Buna ek olarak, bu yaklaşım ventrikül üreten ependim hücreleri ve beyin omurilik sıvısı akış düzlemsel kutuplaşma incelemek için kullanılır olmuştur. Yetişkin sinir kök hücrelerinin bölgesel belirtilen heterojen bir nüfus olduğunu son kanıtlar, wholemount yaklaşım muhtemelen bu kök hücre nişinin organizasyon ve Parselasyon anlamak için önemli bir araç olacaktır.

Watch this Scientific Journal Video about The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow at JoVE.com

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

Lateral ventrikül duvarları en büyük yetişkin memelilerin beyin germinal bölge içerir. Geleneksel olarak, bu bölgedeki nöron çalışmaları klasik histolojik analiz için kesit alma teknikleri dayanıyordu. Burada, bu germinal bölgenin kapsamlı, en-yüz görünümü sağlar wholemount tekniği, alternatif bir yaklaşım sunuyoruz.

En yüze Subventricular Bölge: Wholemount Boyama ve ependim Akış

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an ...

the-subventricular-zone-en-face-wholemount-staining-and-ependymal-flow

Research

JoVE Journal

Neuroscience

41.7K Görüntüleme.  University of California, San Francisco - UCSF. Lateral ventrikül duvarları en büyük yetişkin memelilerin beyin germinal bölge içerir. Geleneksel olarak, bu bölgedeki nöron çalışmaları klasik histolojik analiz için kesit alma teknikleri dayanıyordu. Burada, bu germinal bölgenin kapsamlı, en-yüz görünümü sağlar wholemount tekniği, alternatif bir yaklaşım sunuyoruz.

Video: The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its relatively uniform cytoarchitecture is a complex array of parasagittal domains of gene and protein expression. The molecular compartmentalization of the cerebellum is mirrored by the anatomical and functional organization of afferent fibers. To fully appreciate the complexity of cerebellar organization we previously refined a wholemount staining approach for high throughput analysis of patterning defects in the mouse cerebellum. This protocol describes in detail the reagents, tools, and practical steps that are useful to successfully reveal protein expression patterns in the adult mouse cerebellum by using wholemount immunostaining. The steps highlighted here demonstrate the utility of this method using the expression of zebrinII/aldolaseC as an example of how the fine topography of the brain can be revealed in its native three-dimensional conformation. Also described are adaptations to the protocol that allow for the visualization of protein expression in afferent projections and large cerebella for comparative studies of molecular topography. To illustrate these applications, data from afferent staining of the rat cerebellum are included.

Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.

Revealing Kompleksi Beyin Topografya Wholemount İmmünohistokimya

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its ...

Nörobilim

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

Kalsiyum Görüntüleme için akut subventricular dilimi Dilimleri hazırlanması

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

Yenidoğan subventricular Bölge Elektroporasyon

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

Wholemount Sıçan Retina Immunohistochemical ve Kalsiyum Görüntüleme Yöntemleri

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

Fare Beyin lateral ventrikül Ependimal Cilia Canlı Görüntüleme

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

Hücre Yetişkin Fare subventricular Bölgesinden Nöral Kök ve Progenitör Hücreler Sıralama ve Hücre Döngüsü Dynamics Canlı görüntüleme

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

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

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

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

Fare Subcallosal Zone İzolasyon ve Yetişkin Nöral Kök Hücre Kültürü

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

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

Tek Bir Kemirgen Beyin Örneklerinde İnme Sonrası Serebral Ödem, Enfarktüs Bölgesi ve Kan-Beyin Bariyeri Kırılımının Ölçülmesi

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

Cryo-section Dissection of the Adult Subependymal Zone for Accurate and Deep Quantitative Proteome Analysis

The subependymal neurogenic niche consists of a paraventricular ribbon of the lateral ventricular wall of the lateral ventricle. The subependymal zone (SEZ) is a thin and distinct region exposed to the ventricles and cerebrospinal fluid. The isolation of this niche allows the analysis of a neurogenic stem cell microenvironment. However, extraction of small tissues for proteome analysis&#160;is challenging, especially for the maintenance of considerable measurement depth and the achievement of reliable robustness. A new method termed cryo-section-dissection (CSD), combining high precision with minimal tissue perturbation, was developed to address these challenges. The method is compatible with state-of-the-art mass spectrometry (MS) methods that allow the detection of low-abundant niche regulators. This study compared the CSD and its proteome data to the method and data obtained by laser-capture-microdissection (LCM) and a standard wholemount dissection. The CSD method resulted in twice the quantification depth in less than half the preparation time compared to the LCM and simultaneously clearly outperformed the dissection precision of the wholemount dissection. Hence, CSD is a superior method for collecting the SEZ for proteome analysis.

Cryo-section-dissection allows fresh, frozen preparation of the largest neurogenic niche in the murine brain for deep quantitative proteome analysis. The method is precise, efficient, and causes minimal tissue perturbation. Therefore, it is ideally suited for studying the molecular microenvironment of this niche, as well as other organs, regions, and species.

Doğru ve Derin Nicel Proteom Analizi için Yetişkin Subependymal Bölgesinin Kriyo-kesit Diseksiyonu

The subependymal neurogenic niche consists of a paraventricular ribbon of the lateral ventricular wall of the lateral ventricle. The subependymal zone ...

<meta charset="utf8"></head><body>Murin Subventriküler Bölgesinden İzole Edilen NSC'lerin Etkin Nükleofeksiyonu

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

<meta charset="utf8"></head><body>Yazar Spotlight: Murin Subventriküler Bölgeden Türetilmiş Nöral Kök Hücre Kültürlerinde Yüksek Verimli Gen İletimi için Geliştirilmiş Nükleofeksiyon

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

Erişkin Murin Subventriküler Bölgesinden Nöral Kök Hücrelerin İzolasyonu, Genişlemesi ve Nükleofeksiyonu

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...