Name: generation of single-cell suspensions from mouse neural tissue
Uploaded: 2009-07-07T19:00:00.000+00:00
Duration: 11 min 17 s
Description: Watch this Scientific Journal Video about Generation of Single-Cell Suspensions from Mouse Neural Tissue at JoVE.com

無菌条件下で動作するように、それは層流フード内のすべての手順を実行することをお勧めします。 1。材料<ul><li>神経組織解離キット（P）（コンポーネント：解決策1,2、3、4、記憶バッファー） </li><li>プレセパレーションフィルター</li><li> （オプション）ミエリンの除去ビーズ</li><li> β-メルカプトエタノール</li><li> gentleMACS Dissociator </li><li> Cチューブ</li><li> MACSmixチューブローテーター</li><li> HBSS（W / O） </li><li> HBSS（W） </li></ul><ol><li>プロトコルを開始する前に以下のソリューションを準備します。 <ol><li>二価カチオンのないハンクスバッファード塩類溶液のCa 2 +又はMg 2 +（HBSS（W / O） </li><li> HBSS、標準、すなわちカルシウム2 +およびMg 2 +（HBSS（W） </li></ol></li><li>その後のアプリケーションへの関心の抗原エピトープに応じて、パパインベースの神経組織解離キット（P）またはトリプシンベースの神経組織解離キット（T）のどちらかを使用してください。 </li><li>神経組織解離キットから次の解決策を準備します。 <ol><li>解決策2：0.067 mMのβ-メルカプトエタノールを加える</li><li>ソリューション4：0.7 mlの記憶バッファー（キットに付属）で粉体を溶解、静かに（ボルテックスしないでください）​​ミックス</li><li>酵素ミックス1：Cチューブと10〜15分間インキュベートピペット1.9mLの溶液2と50μlの溶液1（キットからの両方）で37℃これは、脳組織の400mgを解離するのに十分です。 </li></ol></li></ol> 2。神経組織を解離<ol><li> 1 mlのHBSSを（W / O）を含むチューブで脳組織を秤量</li><li>予熱酵素ミックス1 1950μLを含むCチューブにマウスの脳を転送する。 （注：このプロトコルは≤400 mgのために書かれていますが、ソリューションのボリュームがCチューブあたりの脳組織の1600 mgまで対応するようにスケーリングすることができます） </li><li> gentleMACSのDissociatorと、実行プログラム&quot;m_brain_01&quot;にCチューブを置きます。 </li><li> 37℃で15分間回転（MACSmixチューブローテーターを用いて、4回転）でインキュベート℃に</li><li> gentleMACSのDissociatorと、実行プログラム&quot;m_brain_02&quot;にCチューブを置きます。 </li><li>解離のステップの間に静かに解決策3とソリューション4を10μl20μlを混合することによって組織の400 mg当たりの酵素ミックス2の30μlを用意する。 </li><li>セプタムシールキャップを介してCチューブに酵素ミックス2を添加し、ボルテックスすることなく静かに混ぜる。 </li><li> 37℃で10分間回転でCチューブをインキュベートしますインキュベーターでC。 </li><li> gentleMACSのDissociatorと、実行プログラム&quot;m_brain_03&quot;にCチューブを置きます。 </li><li> 37℃で10分間回転でCチューブをインキュベートしますインキュベーターでC。 </li><li>チューブの下部に表示されているサンプルを収集するためにスピンダウンする。 </li></ol> 3。ろ過<ol><li>目的の細胞への通過を許可するフィルタが十分な大きさを選択します。例えば、プルキンエ細胞が30μmのフィルターを通過するには大きすぎるが、プロミニン- 1 +細胞はなります。 </li><li>細胞を除去するsuitable1000μlをピペットを使用するには、セプタムシールキャップを介してCチューブを形成し、15 mlのコレクションチューブにプレセパレーションフィルターに適用します。 （注：より大きなサンプルサイズが50 mlのコレクションチューブを使用するため）。 </li><li> HBSS（w）の10mlでフィルターを洗浄してください。 </li><li>室温で10分間、300 xgでフィルタリングされた細胞を遠心分離します。 </li><li>その後のアプリケーションのための選択のあなたの培地または緩衝液の上澄みを再懸濁します。 </li><li>ミエリンの残骸を削除するには、ミエリンの除去のビーズを使用してください。 </li></ol> 4。代表的な結果：図1-3を参照してください<img src="/files/ftp_upload/1267/Fig_1.png" alt="図1" /> 図。 gentleMACS Dissociatorと1脳の解離は、フローサイトメトリー解析で示すように、97％生存細胞をもたらした。 <img src="/files/ftp_upload/1267/1267fig2.png" alt="図2" /> 図。ニューロスフェア形成の2ライト顕微鏡画像は、磁気細胞選別後MACSでの栽培の7日後に抗プロミニン- 1 MicroBeadsを使用して® NeuroMediumはMACSサプリメントB27 PLUSで補足。細胞は、神経組織解離キット（P）を使用して、CD1マウスの脳（P3）から調製した。 <img src="/files/ftp_upload/1267/1267fig4.jpg" alt="図3.5" /> 図。単細胞懸濁液の3ミエリンの破片は、かなりの細胞分離を損なう、とミエリンの除去ビーズによるミエリン破片の除去は、効率の細胞分離を向上させます。抗プロミニン- 1 MicroBeadsを用いてMACS分離のために、P22マウス脳では神経組織解離キット（P）を用いて解離。単一細胞の懸濁液からの細胞を直接分離のために使用された、またはミエリン削除ビーズを使用して枯渇してミエリンに提出された。ミエリンの除去なしとの試料からの分離を比較すると、純度が以前のミエリンの除去と試料のために高いことを示しています。

<ol>
	<li>Hardt, O., Scholz, C., K&#252;sters, D., Yanagawa, Y., Pennartz, S., Cremer, H., Bosio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+analysis+defines+differences+between+region-specific+GABAergic+neurons.">Gene expression analysis defines differences between region-specific GABAergic neurons.</a> Mol Cell Neurosci. 39, 418-428 (2008).</li><li>Lee, J. K., McCoy, M. K., Harms, A. S., Ruhn, K. A., Gold, S. J., Tansey, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulator+of+G-protein+signaling+10+promotes+dopaminergic+neuron+survival+via+regulation+of+the+microglial+inflammatory+response.">Regulator of G-protein signaling 10 promotes dopaminergic neuron survival via regulation of the microglial inflammatory response.</a> J Neurosci. 28, 8517-8528 (2008).</li><li>K&#246;rnyei, Z., G&#243;cza, E., R&#252;hl, R., Orsolits, B., V&#246;r&#246;s, E., Szab&#243;, B., V&#225;govits, B., Madar&#225;sz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astroglia-derived+retinoic+acid+is+a+key+factor+in+glia-induced+neurogenesis.">Astroglia-derived retinoic acid is a key factor in glia-induced neurogenesis.</a> FASEB J. 21, 2496-2509 (2007).</li><li>Meylan, F., Davidson, T. S., Kahle, E., Kinder, M., Acharya, K., Jankovic, D., Bundoc, V., Hodges, M., Shevach, E. M., Keane-Myers, A., Wang, E. C., Siegel, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TNF-family+receptor+DR3+is+essential+for+diverse+T+cell-mediated+inflammatory+diseases.">The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases.</a> Immunity. 29, 79-89 (2008).</li><li>Skog, J., W&#252;rdinger, T., van Rijn, S., Meijer, D. H., Gainche, L., Sena-Esteves, M., Curry, W. T., Carter, B. S., Krichevsky, A. M., Breakefield, X. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioblastoma+microvesicles+transport+RNA+and+proteins+that+promote+tumour+growth+and+provide+diagnostic+biomarkers.">Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers.</a> Nat Cell Biol. 10, 1470-1476 (2008).</li></ol>

著者らは、ミルテニーバイオテク社、ドイツの従業員です。

gentleMACS Dissociatorは閉鎖系での神経組織から単細胞懸濁液の標準化の準備を容易にします。神経組織の解離のキットはimmunostainingsと免疫磁気細胞分離1から5のような、さらにアプリケーションに必要な抗原のエピトープを保持するために最適化されています。このプロトコルでは、97％生存細胞に降伏、gentleMACS Dissociatorと神経組織解離キット（P）を使用してマウスの脳の穏やかな酵素的解離を示す。宿主組織の年齢に応じて、5 × 10 6と1 × 10 7細胞、間に神経組織の収量100 mgの。標的プロミニン- 1 +細胞は、抗プロミニン- 1 MicroBeadsを用いて単離し、その後、文化に取り込まれた。プロトコルは、マウス&gt; P7由来の神経組織で作業するときミエリン除去ビーズの追加使用でさらに効果的であることが示されている。

<table><tbody><tr><td>C Tubes</td><td>Miltenyi Biotec</td><td>Order no. 130-093-237</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx&lt;/a&gt;</td></tr><tr><td>gentleMACS&amp;#8482; Starting Kit</td><td>Miltenyi Biotec</td><td>Order no. 130-093-235</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx&lt;/a&gt;</td></tr><tr><td>MACSmix&amp;#8482; Tube Rotator</td><td>Miltenyi Biotec</td><td>Order no. 130-090-753</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_34_251_MACSmix_Tube_Rotator.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_34_251_MACSmix_Tube_Rotator.aspx&lt;/a&gt;</td></tr><tr><td>Neural Tissue Dissociation Kit (P)</td><td>Miltenyi Biotec</td><td>Order no. 130-092-628</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_712_656_Neural_Tissue_Dissociation_Kits.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_712_656_Neural_Tissue_Dissociation_Kits.aspx&lt;/a&gt;</td></tr><tr><td>Pre-Separation Filters</td><td>Miltenyi Biotec</td><td>Order no. 130-041-407</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_34_247_Pre_Separation_Filters.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_34_247_Pre_Separation_Filters.aspx&lt;/a&gt;</td></tr><tr><td>Anti-Prominin-1 MicroBeads</td><td>Miltenyi Biotec</td><td>Order no. 130-092-333</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_654_524_Anti_Prominin_1_MicroBeads.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_654_524_Anti_Prominin_1_MicroBeads.aspx&lt;/a&gt;</td></tr><tr><td>Anti-Prominin-1-APC</td><td>Miltenyi Biotec</td><td>Order no. 130-092-335</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_599_525_Anti_Prominin_1.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_599_525_Anti_Prominin_1.aspx&lt;/a&gt;</td></tr><tr><td>Myelin Removal Beads</td><td>Miltenyi Biotec</td><td>Order no. 130-094-544</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_712_688_Myelin_Removal_Beads.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_712_688_Myelin_Removal_Beads.aspx&lt;/a&gt;</td></tr><tr><td>Art 1000 Reach Pipet Tips </td><td>Molecular BioProducts</td><td>Order no. 2079</td><td>&lt;a href=&quot;http://www.mbpinc.com/ASP/products.aspx?query_TipID=61&quot;&gt;http://www.mbpinc.com/ASP/products.aspx?query_TipID=61&lt;/a&gt;&lt;br/&gt;&lt;a href=&quot;http://www.mbpinc.com/html/products/art/display.html&quot;&gt;http://www.mbpinc.com/html/products/art/display.html&lt;/a&gt;</td></tr><tr><td>Neural Tissue Dissociation Kit (T)</td><td>Miltenyi Biotec</td><td>Order no. 130-093-231</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_712_656_Neural_Tissue_Dissociation_Kits.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_712_656_Neural_Tissue_Dissociation_Kits.aspx&lt;/a&gt;</td></tr><tr><td>Propidium Iodide Solution</td><td>Miltenyi Biotec</td><td>Order no. 130-093-233</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_337_744_Propidium_Iodide_Solution.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_337_744_Propidium_Iodide_Solution.aspx&lt;/a&gt;</td></tr></tbody></table>

generation of single-cell suspensions from mouse neural tissue

神経系の中で、神経細胞およびグリア細胞の数百種類が記載されている。脳または脊髄内の各特定の細胞型は、細胞表面分子、またはそれを特定して特徴付けることができるような分子決定因子、のレパートリーを持っています。現在のところ、堅固な細胞の同定と分離技術は、同時に特徴的な表面タンパク質の細胞死と破壊を制限しながら、生成される単一セルの準備が必要です。抗原のエピトープを保持し、細胞の損失を制限しながら、トリプシンやパパインベースの解離キットと組み合わせて使用​​されるgentleMACS Dissociatorは、効果的かつ穏やかに脳組織を切り離すことができます。単一の細胞懸濁液の標準化された製剤は、Cチューブと最適化された、プリセットgentleMACSプログラムを使用して実現されます。一度生成された、単細胞懸濁液は、神経前駆細胞を識別する、またはミエリンの除去のビーズを使用してさらに精製した抗プロミニン- 1マイクロビーズのようなモノクローナルコンジュゲートで治療することができます。

Watch this Scientific Journal Video about Generation of Single-Cell Suspensions from Mouse Neural Tissue at JoVE.com

Generation of Single-Cell Suspensions from Mouse Neural Tissue

特定の組織型から解離し細胞が組織aggitationのための特定のパラメータは、実行可能な、培養可能な細胞の大量を取得する必要があります。ミルテニーgentleMACS Dissociatorは、シンプルで実用的なプロトコルを使用してこのタスクを最適化します。本書ではnerual組織でこの装置の使用は説明されています。

マウス神経組織から単細胞懸濁液の生成

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

generation-of-single-cell-suspensions-from-mouse-neural-tissue

Research

JoVE Journal

Biology

22.5K ビュー.  Miltenyi Biotec,GmbH.  中文 日本語 

ビデオ: Generation of Single-Cell Suspensions from Mouse Neural Tissue

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

マウス神経幹細胞の前駆体の文化

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

生物学

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.

ニューロスフェアのアッセイを使用してマウス胎児神経幹細胞培養の確立

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 ...

神経科学

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

廃棄されたヒト胎児脳組織から神経幹細胞の生成

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

マウスを一回、二つの文化：個々のマウスの二つの神経原性のゾーンから成体神経幹細胞の単離および培養

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

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.

マウス梁下ゾーンから成体神経幹細胞の単離および培養

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

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

マウス萌芽期の神経幹細胞の分離と培養

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

<meta charset="utf8"></head><body>マウス脳室内領域から単離されたNSCの効率的なヌクレオフェクション

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>著者スポットライト:マウス脳室下ゾーン由来神経幹細胞培養における高効率遺伝子導入のためのヌクレオフェクションの改善

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.

成体マウス脳室下領域からの神経幹細胞の単離、増殖、およびヌクレオフェクション

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 ...

マウスの脳および頭蓋骨骨髄からの単一細胞懸濁液の取得

Isolating Immune Cells from Mouse Brain and Skull

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only in the brain, but also in the skull. A key step toward analyzing changes in immune cell populations in both the brain and skull bone marrow after brain damage (e.g., stroke) is to obtain sufficient numbers of high-quality immune cells for downstream analyses. Here, two optimized protocols are provided for isolating immune cells from the brain and skull bone marrow. The advantages of both protocols are reflected in their simplicity, speed, and efficacy in yielding a large quantity of viable immune cells. These cells may be suitable for a range of downstream applications, such as cell sorting, flow cytometry, and transcriptomic analysis. To demonstrate the effectiveness of the protocols, immunophenotyping experiments were performed on stroke brains and normal brain skull bone marrow using flow cytometry analysis, and the results aligned with findings from published studies.

著者スポットライト:脳虚血後の複雑な免疫応答の調査

To investigate the immune response to brain disorders, one common approach is to analyze changes in immune cells. Here, two simple and effective protocols are provided for isolating immune cells from murine brain tissue and skull bone marrow.

マウスの脳と頭蓋骨からの免疫細胞の単離

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only ...

マウス神経組織から単細胞懸濁液の生成

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ニューロスフェアのアッセイを使用してマウス胎児神経幹細胞培養の確立

廃棄されたヒト胎児脳組織から神経幹細胞の生成

マウスを一回、二つの文化：個々のマウスの二つの神経原性のゾーンから成体神経幹細胞の単離および培養

マウス梁下ゾーンから成体神経幹細胞の単離および培養

マウス萌芽期の神経幹細胞の分離と培養

成体マウス脳室下領域からの神経幹細胞の単離、増殖、およびヌクレオフェクション

成体マウス脳室下領域からの神経幹細胞の単離、増殖、およびヌクレオフェクション

成体マウス脳室下領域からの神経幹細胞の単離、増殖、およびヌクレオフェクション

マウスの脳と頭蓋骨からの免疫細胞の単離