我々は最初のプロトコルを妊娠中レナータ佐野の貢献を認める。 Ad&#39;A。遺伝学と遺伝子治療に寄附チルドレン（JFC）のために宝石を保持しています。この作品は、NIHの助成金GM60905、DK52025とCA021764、アメリカレバノンシリア関連慈善団体（ALSAC）によって部分的に資金を供給された。

<html>以下のプロトコールは、マウスの脳からMAMSや宝石の単離および精製のために意図されているミトコンドリア、MAMSや宝石の分離に必要なソリューション原油ミトコンドリアの準備を取得するための分別： ソリューション：0.32Mスクロース、1mMのNaHCO 3を 、1mMのMgCl 2、0.5 mMのCaCl 2 +プロテアーゼ阻害剤（必要に応じて、新鮮追加） B液：0.32Mスクロース、1mMのNaHCO 3を +プロテアーゼ阻害剤（必要に応じて、新鮮追加） MAMSアイソレーション： 分離培地：250mMのマンニトール、5mMのHEPES緩衝液（pH7.4）、0.5mMのEGTA、0.1％のBSA 勾配バッファ：225 mMのマンニトール、25mMのHEPES緩衝液pH7.5、1mMのEGTA、0.1％のBSA（最終濃度） GEMSの分離： コンテンツ&quot;&gt; GEMの抽出バッファー：25mMの HEPES、pH7.5の0.15MのNaCl、1％トリトンX-100 +プロテアーゼ阻害剤（必要に応じて、新鮮追加） GEMの可溶化緩衝液：50mMのトリス-HCl、pH 8.8、5mMのEDTA、1％SDS +プロテアーゼ阻害剤（必要に応じて、新鮮追加）。 1。最初のステップ細胞下分画：核の除去、Unlysed細胞および細胞破片<ol><li> CO 2のチャンバー内でマウスを安楽死させる。 </li><li>すぐに、脳を除去半減、氷の上に2 mlチューブに置き、重量を量る。 </li></ol> 注：手順全体を通して脳の半分を分離します。以下は、単一の脳の半分の治療に適用されます。 <ol start="3"><li> 1ミリリットル冷たい溶液Aが大きいクリアランス乳棒の合計15ストロークでホモジナイズ含む予冷2ミリリットルのガラス製ダウンス組織グラインダーで行わハーフ脳。 </li><li>氷の上で、15mlファルコンチューブに移し、希釈10ボリュームw / vの、 例えば 0.225グラム〜2.25 mlまでホモジネート。 </li><li> 4℃で10分のために1400 xgで遠心し</li><li>上清を慎重に除去し、氷の上に、30 mlの丸底ガラス遠心管に移す。ペレットを乱さないように注意してください。 </li><li> ソリューションの同じ10巻でペレットを再懸濁します。小さなクリアランス乳棒、一度に1ミリリットルの3から6ストロークで、同じグラインダーでホモジナイズする。 </li><li> 4℃で10分間、710×gでフレッシュ15mlファルコンチューブおよび遠心℃に移す核および細胞破片のペレットでこの結果。 </li><li>慎重にステップ1.6で保存した上清を用いて、上清とプールを削除します。 </li></ol> 2。第二ステップ：粗ミトコンドリア単離<ol><li> 4℃で10分のために13800×gで遠心分離し、上清を</li><li>氷の上で、超クリアベックマン遠心チューブに上清を移し、 </li><li>ペレットを再懸濁し10倍量のソリューションインチ小さなクリアランス乳棒、一度に1ミリリットルの3から6ストロークで、同じグラインダーでホモジナイズする。 </li><li>ステップ2.1のように遠心分離します。 </li><li>ステップ2.2から2.4を繰り返します。 </li><li>得られたペレットは、濃縮されたミトコンドリア画分である。プールの上清（細胞質ゾルとER）、パラフィルムでカバーし、氷上で保存する。 </li><li>新鮮なホモジナイザーで、B液の4.8ミリリットル/グラム（gは元脳重量と呼ばれます）で、小さな隙間乳棒の6ストロークでペレットを再懸濁します。 </li><li>ガラスパスツールピペットを用いて、超クリアベックマン遠心管に不連続ショ糖密度勾配を作成し、次のようにチューブの底に続いて追加して、慎重に、気泡が形成されない確実： Resupendedペレット（0.32Mスクロース） 3ミリリットル850 mMスクロース（1ミリメートルのNaHCO 3） 3ミリリットル1 Mスクロース（1ミリメートルのNaHCO 3） 3ミリリットル1.2 Mショ糖（1mMの炭酸水素ナトリウムで3） </li><li> 4℃で2時間、82500×gで遠心分離し、グラデーションの終わりに得られた分離は、3つのバンドとペレット生成されます： - 、2）小胞体、ゴルジ体、原形質膜（0.85 M - 1 Mインターフェイス）0.85 Mインターフェイス）1）ミエリン及び他の膜汚染物質を（0.32 M; 3）シナプトソーム（1 - M 1.2 Mインターフェイス）、4）ミトコンドリア原油（ペレット）は、その後の精製工程のために使用される</li></ol> 3。ミトコンドリア関連ER膜の分離、MAMS <ol><li>新たに追加されたプロテアーゼ阻害剤を含む2 mlの分離培地では、1つの脳の半分から新たに単離したミトコンドリア原油を再懸濁します。 </li><li> 勾配緩衝8半脳mlあたり、および超クリアベックマン遠心チューブ内の場所で30％パーコール勾配を準備します 。 </li></ol> * 注：（1.43倍）勾配バッファはもっと集中してくださいdの ​​後に適切な最終濃度を達成するために30％パーコールをiluting。 <ol start="3"><li>準備されたグラデーションの上の層ミトコンドリア懸濁液は徐々に任意の気泡を防ぐことができます。 </li><li> 30分、4℃で95000×gで遠心分離し、 </li><li><ol class="lroman"><li>ガラスパスツールピペットを持つ最初の重質留分（下部バンド）を削除し、新鮮な丸底ガラス管に移し、氷の上で。 </li><li>別のガラスパスツールピペットを用いて、第二の光フラクション（上部バンド）を取り外して、氷の上で、別々の新鮮な丸底ガラス管に移す。 </li></ol></li><li> 4℃で10分間6300 xgで℃で10 mlの分離培地と遠心分離機でステップ3.5で回収した画分の両方を薄める</li><li>ステップ3.6で得られた重質留分からの上清を捨て、ステップ3.6のように、再び別の10 mlの分離培地と遠心でペレットを再懸濁します。得られたペレットは、 純粋なミトコンドリア画分となります。 </li><li>ライトフラクショナルから上清を移するには、氷の上で、超クリアベックマン遠心チューブにステップ3.6で得られた。ペレットを廃棄します。 </li><li> 1時間、4℃、100,000×gでチューブと遠心分離機を埋めるために、ステップ3.8で得られた上清に十分な分離培地を追加得られたペレットは、MAMS分数となります。上清を慎重に除去して廃棄します。 </li></ol> 注：このステップで、あなたも1時間、4、100,000 xgで遠心あり℃ステップ2.6で保存した上清。ペレットは、ER分数であるとサイトゾル画分を上澄みます。 4。スフィンゴ糖脂質に富むマイクロドメインの抽出、GEMS <ol><li>氷上で20分間のExtraction Bufferを500μl-1mlに純粋なミトコンドリアおよび/またはMAM分画を溶解します。 </li><li> 4℃で2分間15300×gでライセートを遠心℃、 </li><li>上清（トリトンX-100可溶性物質）および再遠心ペレットを集める2分間残っている水溶性物質を除去します。 （トリトンX-100ミトコンドリア抽出および/またはTriton X-100抽出MAMS）。 </li><li>バッファーを可溶化にペレットを可溶化する。この可溶化された材料は、GEM分数を表し、さらなる分析のために使用することができます。 </li></ol>

<ol>
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細胞内膜間または細胞小器官や細胞の細胞膜との間の接触部位は、基本的な細胞プロセスのためのダイナミックシグナリングプラットフォームを表します。生理学的および病理学の両方の条件下でその機能と組成の正確な特性評価には信頼性と再現性の高い精製プロトコルを必要とします。ここで詳述した方法は、特に成体マウスの脳からMAMSとそれらに対応する宝石の単離および精製のため...

<table border="1"><tbody><tr><td> 試薬/材料の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td></td><td></td><td></td><td> 試薬 </td></tr><tr><td></td><td></td><td></td><td> 分別 </td></tr><tr><td>スクロース</td><td>フィッシャー·サイエンティフィック</td><td> S5-500 </td><td></td></tr><tr><td>重炭酸ナトリウム</td><td>シグマアルドリッチ</td><td> S-5761 </td><td></td></tr><tr><td>塩化マグネシウム六水和物</td><td>フィッシャー·サイエンティフィック</td><td> BP214-500 </td><td></td></tr><tr><td>塩化カルシウム二水和物</td><td>シグマアルドリッチ</td><td> C-5080 </td><td></td></tr><tr><td></td><td></td><td></td><td> MAM </td></tr><tr><td> D-マンニトール</td><td>シグマアルドリッチ</td><td> M9546-250G </td><td></td></tr><tr><td>ヘペス</td><td>フィッシャー·サイエンティフィック</td><td> BP310-500 </td><td></td></tr><tr><td> EGTA </td><td>シグマアルドリッチ</td><td> E4378-250G </td><td></td></tr><tr><td> BSA、フラクションV、熱ショック、凍結乾燥</td><td>ロッシュ</td><td> 03-116-964-001 </td><td></td></tr><tr><td>パーコール</td><td> GE </td><td> 17-0891-02 </td><td></td></tr><tr><td></td><td></td><td></td><td> GEM </td></tr><tr><td>トリトンX-100 </td><td>シグマアルドリッチ</td><td> T9284-500ミリリットル</td><td></td></tr><tr><td>塩化ナトリウム</td><td>フィッシャー·サイエンティフィック</td><td> S271-3 </td><td></td></tr><tr><td>トリス塩基</td><td>ロッシュ</td><td> 03-118-142-001 </td><td></td></tr><tr><td>塩酸</td><td>フィッシャー·サイエンティフィック</td><td> A144S-500 </td><td></td></tr&gt; <tr><td> EDTAを</td><td>フィッシャー·サイエンティフィック</td><td> BP120-500 </td><td></td></tr><tr><td>ドデシル硫酸ナトリウム（SDS） </td><td>フィッシャー·サイエンティフィック</td><td> BP166-500 </td><td></td></tr><tr><td></td><td></td><td></td><td> 共通の </td></tr><tr><td>完全プロテアーゼ阻害剤錠、EDTAフリー</td><td>ロッシュ</td><td> 11-873-580-001 </td><td></td></tr><tr><td></td><td></td><td></td><td> 機器 </td></tr><tr><td> 2ミリリットルDouceの全ガラスティッシュグラインダー</td><td>キンブルチェイス</td><td> 885300-0002 </td><td></td></tr><tr><td> 15mlのポリプロピレンコニカル遠心管、BDファルコン</td><td> BD </td><td> 352097 </td><td></td></tr><tr><td> 30ミリリットル丸底ガラス遠心管</td><td>キンブルチェイス</td><td> 45500から30 </td><td></td></tr><tr><td> 15ミリリットル丸底ガラス遠心フーガチューブ</td><td>キンブルチェイス</td><td> 45500から15 </td><td></td></tr><tr><td>超遠心管、超クリア、薄肉、14x89ミリメートル</td><td>ベックマン·コールター- </td><td> 344059 </td><td></td></tr><tr><td>パラフィルム</td><td>コー​​ル - パーマー</td><td> PM996 </td><td></td></tr><tr><td>使い捨てのホウケイ酸ガラスパスツールピペット、9 &quot; </td><td>フィッシャー·サイエンティフィック</td><td> 13から678-20C </td><td></td></tr></tbody></table>

mitochondria-associated er membranes (mams) and glycosphingolipid enriched microdomains (gems): isolation from mouse brain

細胞内小器官は、細胞特異的内因性および外因性の刺激にさらされる形状および組成を変化させると非常にダイナミックな構造です。その膜は、しばしば、シグナル伝達分子や膜成分1,2,3,4の交換のためのハブとなって定義されている接触部位で並置されている。 IP3は敏感なCa 2 +チャネルの開口部には小胞体（ER）とミトコンドリアの間に形成されている間オルガネラ膜マイクロドメインは、ミトコンドリア関連する- ER膜またはMAMS 4,5,6として知られている。タンパク質/脂質組成およびこれらの膜の接触部位の生化学的特性は、広範囲に、特に細胞内Ca 2 + 4,5,6の調節におけるそれらの役割に関連して研究されてきた。 ERは、細胞内Ca 2 +の主な店舗となっており、この容量で下流のCa 2の細胞プロセスの無数+シグナル、税込を調節翻訳後のタンパク質の折りたたみ及びタンパク質maturation7をuding。ミトコンドリアは、その一方で、それによって下流のCa 2 +のアンバランス4,8のアポトーシス経路の開始を防止する細胞内Ca 2 +濃度をバッファリングすることにより、Ca 2 +の恒常性を維持する。 MAMSの動的な性質は、それらの理想的なサイトは、Ca 2 +シグナル伝達とミトコンドリアのCa 2 +濃度、脂質生合成と輸送、エネルギー代謝と細胞生存4,9,10,11,12の調節など、基本的な細胞メカニズムを解剖することができます。いくつかのプロトコルが、肝組織および培養細胞13,14からこれらのマイクロドメインの精製の ​​ために記載されている。 アカウントに以前に公開されている方法を取って、我々は、成体マウスの脳からミトコンドリアとMAMSを単離するためのプロトコルを適応している。この手順に、我々はどの電子すなわち余分な精製工程、トリトンX100の抽出を追加しましたnables MAMSのスフィンゴ糖脂質に富んだマイクロドメイン（GEM）の画分の単離。これらのGEMの製剤は、原形質膜または他の細胞内膜に由来し、カベオラや脂質ラフトといくつかのタンパク質成分を共有しており、受容体タンパク質のクラスタリングのために、タンパク質-タンパク質相互作用4,15の集まるポイントとして機能するように提案されている。

このプロトコルを使用しているとの経験に基づいて、我々は安全MAMS、宝石、マウス脳のミトコンドリア画分の単離および精製のためにそれをお勧めすることができます。概説された手順は、非常に再現性があり、一貫性があります。 図1に我々は、パーコール勾配（ステップ3.4）上の方法で純粋なミトコンドリアとMAMS層の代表的なイメージを示しています。 MAMのフラクションは...

Watch this Scientific Journal Video about Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain at JoVE.com

Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

この手順では、成体マウスの脳ミトコンドリア関連のER膜またはMAMSとMAMSとミトコンドリア調製物からスフィンゴ糖脂質に富むマイクロドメイン画分から単離する方法を示しています。

ミトコンドリア関連のER膜（MAMS）とスフィンゴ糖脂質マイクロドメインエンリッチド（GEMS）：マウス脳から単離

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic ...

mitochondria-associated-er-membranes-mams-glycosphingolipid-enriched

Research

JoVE Journal

Neuroscience

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

24.3K ビュー.  St Jude Children's Research Hospital. この手順では、成体マウスの脳ミトコンドリア関連のER膜またはMAMSとMAMSとミトコンドリア調製物からスフィンゴ糖脂質に富むマイクロドメイン画分から単離する方法を示しています。

ビデオ: Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

出生後のマウス組織から培養オリゴデンドロサイトと共培養Myelinatingニューロン/オリゴデンドロサイトエンリッチドの導出

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

神経科学

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

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

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

胚前脳からの内皮細胞の単離および培養

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

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings.&#160;The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.

We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and memory.

Synapto核メッセンジャータンパク質の活性に依存する核内移行を研究するために海馬スライスからCA1原子力エンリッチ画分の単離

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

胚性齧歯類の脳から単離、プライマリ脳ドーパミンニューロンの文化と長期メンテナンス

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

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 of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

新鮮な人間の脳から脳の毛細血管の分離

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

分離およびゼブラフィッシュ稚魚の脳から神経細胞、大食細胞、ミクログリアの RNA の抽出

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

ディープシングルセルRNAシーケンシングのための1つの成人マウス脳半球からの領域特異的ミクログリアの分離

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Isolation and Expansion of Neurospheres from Postnatal (P1&#8722;3) Mouse Neurogenic Niches

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including proliferation, self-renewal and multipotency. In the postnatal and adult brain, NSPCs are mainly present in two neurogenic niches: the subventricular zone (SVZ) lining the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus (DG). The isolation of the neurogenic niches from postnatal brain allows obtaining a higher amount of NSPCs in culture with a consequent advantage of higher yields. The close contact between cells within each neurosphere creates a microenvironment that may resemble neurogenic niches. Here, we describe, in detail, how to generate SVZ- and DG-derived neurosphere cultures from 1&#8722;3-day-old (P1&#8722;3) mice, as well as passaging, for neurosphere expansion. This is an advantageous approach since the neurosphere assay allows a fast generation of NSPC clones (6&#8722;12 days) and contributes to a significant reduction in the number of animal usage. By plating neurospheres in differentiative conditions, we can obtain a pseudomonolayer of cells composed of NSPCs and differentiated cells of different neural lineages (neurons, astrocytes and oligodendrocytes) allowing the study of the actions of intrinsic or extrinsic factors on NSPC proliferation, differentiation, cell survival and neuritogenesis.

In this article, we describe, in detail, a protocol for the generation of neurosphere cultures from postnatal mouse neural stem cells derived from the main mouse neurogenic niches. Neurospheres are used to identify neural stem cells from brain tissue allowing the estimation of precursor cell numbers. Moreover, these 3D structures can be plated in differentiative conditions, giving rise to neurons, oligodendrocytes and astrocytes, allowing the study of cell fate.

出生後(P1-3)マウス神経起源ニッチからの神経球の分離と拡大

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including ...

Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on &#945;-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson&#8217;s disease, Lewy bodies (LB), is mimicked by the addition of &#945;-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated &#945;-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing &#945;-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-&#945;-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated &#945;-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of &#945;-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.

Here, we present a detailed protocol to study neuronal &#945;-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated &#945;-synuclein aggregates in neurons are induced with pre-formed &#945;-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit &#945;-synuclein accumulation.

原発性胚マウス中脳ドーパミンニューロンにおける前形成線維誘導α-シヌクレイン蓄積の研究

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with ...