我々は彼らのアドバイスや有益なコメントに対してTsirka研究室のメンバーに感謝したいと思います。この作品は、JCNにRB、NSF-3MT IGERTとLTにターナー論文のフェローシップ、およびNSF-3MT IGERTに、12PRE12060489を設定するためにR01NS42168によってサポートされていました。

1。解剖（0日目） <ol><li>低体温とP0-2マウス仔に麻酔を誘導する。組織を消毒するために70％エタノールに浸したキムワイプで子犬の頭を拭いてください。 </li><li> 10センチ培養プレートあたり4仔を用いた、はさみで頭を取り外します。氷冷ハンクスバッファーを含むペトリ皿に頭を置きます。 </li><li>眼窩を通して湾曲したピンセットを使用してヘッドを固定し、慎重に真っ直ぐmicroforcepsと頭蓋骨をカバーする皮膚を除去。 </li><li>パンクをしないか、または皮質を損傷に注意して使用し、ストレートmicroforcepsと頭蓋骨を取り外します。この領域は破棄されるように、最も効果的な方法は、頭の基部に位置している小脳の近く開始除去を開始することです。 </li><li>小さなへらで脳を取り出し、氷冷ハンクス緩衝液を含有する新鮮なペトリ皿に（4）脳を配置。 </li><li> microforcepsを利用し、この時点から、すべてのステップはdisse下で行われているction顕微鏡。脳の腹側から始まる、小脳を保持することによって、組織を固定し、中脳の両側にある2つの小さな切開を行います。皮質は、この地域の中脳をカバーするように、組織を介してすべての道を切らないように注意してください。 </li><li>ゆっくり2皮質から一枚で中脳と小脳をいじめる。 2皮質は、凹面形状を形成する必要があります。 </li><li>さらに解剖のために内側の2つの皮質とオリエント一つの皮質を区切ります。海馬は、観察することが困難になる可能性がありますが、これは大脳皮質の先端に小さな結節として現れる嗅球の反対側に位置しています。 </li><li>三日月形状をしている海馬を、削除します。背側を表示するには、皮質を裏返し。 </li><li>出発点として、嗅球を使用して、すべての髄膜と皮質から嗅球自体を削除します。 </li><li>解剖皮質はCOL 14mlの、15 mlコニカルチューブに配置する必要があります氷の上でDハンクス緩衝生理食塩水。 </li></ol> 2。細胞培養（0日目） <ol><li> 37℃で3時間オートクレーブに水で希釈したポリ-D-リジン（PDL）5μgの/ mLでプレコート10センチ組織培養皿</li><li> PDLを吸引し、オートクレーブ処理水で1回プレートを洗浄する。 20分間組織培養フード内UV下で乾燥したプレート。このステップは、切開プロセスの直前に行​​われるべきである。 </li><li> 4脳からそれぞれ皮質を含むチューブから吸引ハンクスバッファは、1×トリプシン/ EDTA溶液4mlを適用し、℃のインキュベーターで15分間37にP1000先端、および場所管と組織を摩砕。 </li><li>酵素消化を停止するには、チューブあたり完全ミクログリア培地4mlの、ミックスを追加し、5分間1.5K rpmで内容をスピンダウン。 </li><li>上清を吸引し、完全培地4mlで洗浄を繰り返します。マイクロ40を介して完全なミクログリア培地（10％FBS、1％ピルビン酸ナトリウムを含むDMEM、ゲンタマイシン0.08％）、及びフィルタ10ml中の細胞を再懸濁しn個のメッシュセルストレーナー。 </li><li> 37における組織培養インキュベーター10cmの組織培養プレートと場所25.4 1ml当たり8皮質の密度でプレート℃、5％CO 2（アダルトミクログリアプロトコルを参照）。 </li></ol> （3日目） <ol><li>完全ミクログリアメディアとすべての細胞培養皿内のメディアを変更します。 </li></ol> （10日目） <ol><li> 60mmの400マイクロリットル10cmの組織培養プレートの培地と10〜15分間室温（RT）でインキュベートにHBSS（ミクログリアを切り離す）にリドカインを追加します。 </li><li>プレートからメディア/細胞懸濁液を収集し、ハンクのバッファで一回プレートを洗浄する。残りのミクログリアを回復するために洗浄バッファーを収集します。 </li><li> 50μMの最終濃度まで細胞懸濁液に5mMのEDTAを（pH8.0）を追加したり、1/100希釈で。 </li><li> 5分間1,000×gで（1,500 rpm）で細胞懸濁液をスピンダウンし、1％FBSを1ミリリットルDMEM中で再懸濁する。 </li><li> （生細胞数をカウントアダルトミに従ってcroglia 3.1/3.2）、目的の実験密度における組織培養プレートに分割した細胞。約1×10 6ミクログリア4脳から皮質を含む10センチプレートから採取される。免疫については、18ミリメートルカバースリップあたり2.5×10 4細胞の密度が推奨されます。 </li><li>ミクログリア細胞は、少なくとも24時間は完全に使用する前に、その分岐し、安静時の状態に戻すことができます。 </li></ol>プロトコルテキスト：（大人ミクログリア） 1。組織採取<ol><li> 37℃で3時間、ポリ-D-リジン（PDL）でコート組織培養プレート°Cまたは一晩4℃でオートクレーブ処理水で1回使用直前にプレートを洗ってください。 </li><li> CO 2窒息と安楽死が完全であることを確かめることによって、マウスを安楽死させる。 70％エタノールで脊髄エリアを清掃してください。 </li><li>はさみのペアを使用すると、脊髄の上に皮をカットしてから、領域T1からT12に脊髄を切り取るに進みます。カット脊髄の辺の残りの筋肉オフ。 </li><li>徐々にあなたはそっと指先で脊髄を保持としてmicroscissorsを使って脊椎を切った。組織が非常に柔らかいですとして脊髄穿刺しないように注意してください。ゆっくりmicroforcepsを使って脊髄を抽出​​します。 </li><li>氷のように冷たいHBSSを含むペトリ皿で​​水没脊髄。光学顕微鏡を使用すると、microforcepsを使用して、すべての可視の髄膜を除去</li><li>いくつかの断片を生成するのに十分に小さい横断セグメントに脊髄をカット。断片の厚さが効率的な消化を容易にするために、2mm以下であってはならない。 1ミリリットル氷冷HBSSを含む15ミリリットルコニカルチューブに脊髄の部分を転送します。消化工程まで、チューブを氷上に保管してください。 </li></ol> 2。ティッシュの消化<ol><li>脊髄組織を含む15ミリリットルコニカルチューブからHBSSを吸引。組織のどの部分を吸引しないように注意してください。 </li><li>トリプシン（2の1ミリリットルを追加。5 HBSSのG / L照射ブタトリプシンおよび0.2グラム/ L EDTA）、37℃で30分間インキュベートする。 </li><li>消化を停止するにはトリプシンを除去し、酵素消化を停止するために血清含むプライマリミクログリア培地3mlを加える。 </li><li>組織を取り除くために、上下にピペット。 40μmのセルストレーナーを使用して細胞懸濁液をろ過する。 </li></ol> 3。細胞計数とめっき<ol><li>細胞懸濁液及びDAPI溶液を等量混合する。 </li><li>血球計算板を用いて細胞を数える。 </li><li> PDLでコーティングされた35×10ミリメートル皿中5-7×10 5細胞/ mlの密度でプレート。細胞を、35×10 mmディッシュに比べて小さい面積を有する12ウェルプレートで培養した場合であっても低い密度でメッキは、細胞の成長を防止する。 </li></ol>

<ol>
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Neurosci. 26, 1880-1887 (2006).</li><li>Sasmono, R., Oceandy, D., Pollard, J., Tong, W., Pavli, P., Wainwright, B., Ostrowski, M., Himes, S., Hume, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+macrophage+colony-stimulating+factor+receptor-green+fluorescent+protein+transgene+is+expressed+throughout+the+mononuclear+phagocyte+system+of+the+mouse.">A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse.</a> Blood. 101, 1155-1163 (2003).</li><li>Ekdahl, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+activation+-+tuning+and+pruning+adult+neurogenesis.">Microglial activation - tuning and pruning adult neurogenesis.</a> Front Pharmacol. 3, 14 (2012).</li><li>Sierra, A., Gottfried-Blackmore, A., McEwen, B., Bulloch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+derived+from+aging+mice+exhibit+an+altered+inflammatory+profile.">Microglia derived from aging mice exhibit an altered inflammatory profile.</a> Glia. 55, 412-424 (2007).</li></ol>

著者らは、開示することは何もありません。

ミクログリアは、CNSの正常な機能だけでなく、様々な病態に炎症反応を調節する。ミクログリアによる機能性シナプスリモデリングは、正常な脳ホメオスタシス15の維持に関与している。神経性カスケードの間、彼らは、海馬4、16の歯状回から神経前駆細胞の隙間に参加しています。したがって、ミクログリアは、複数の個別の機能を有する全体の開発と成人の広大な帯状を?...

ミクログリアは、最も密接に構造と機能1に周辺マクロファージに類似し、CNSの居住者の免疫細胞である。それは、最近生後ミクログリア細胞は出生後の造血前駆細胞は成人の脳2ミクログリアの源であることを以前の概念をupending、原始骨髄前駆細胞から派生し、胚発生の八日前に生成されていることが実証されている。彼らは、いくつかの神経疾患において重要な役割を果たしているとすぐに炎症誘発性または抗炎症性サイトカイン3を解放することによって感染症やけがに対応することができます。したがって、ミクログリアは、疾患の進行に影響を与えるために操作することができるCNS内のスタンドアロンユニットを包含する。分離するために、堅牢で再現性のある方法と文化新生児または成人のミクログリア細胞の開発は、今後の研究にとって重要である。 ミクログリアは、脳病変の数が重要なプレーヤーであることが知られている。より最近では、リングライトESは海馬1、4の歯状回からミクログリア貪食過剰神経前駆細胞として正常な脳の発達と機能にセルに浮上している。ミクログリアは、そのような5-7 MS、神経因性疼痛、および脊髄損傷などの脊髄に影響するいくつかの神経学的条件をも調節することができる。脊髄ミクログリアは、ローカル環境の違いにおそらく活性化信号8,9に応答して脳ミクログリアと比較して異なって反応する。このようにして培養するインビトロ系で適切を確立し、脊髄ミクログリアを検討することが重要である。新生児ミクログリアは、IFN-γ、またはTNF-10,11さらには、特定の疾患のコンテキストでミクログリアを研究するために、成人の細胞を使用する必要性を強調してin vitro刺激後に成体細胞と比較して炎症性サイトカインの窒素酸化物のかなり多くを生成する。 我々は、CUにラボで採用するプロトコルlture新生児ミクログリアは、細胞培養フラスコ12の表面から小膠細胞を除去する目的で、混合膠細胞培養の揺れ利用する最近の方法の変形例である。我々はまた、最初のイップら 13で記述されたプロトコルに基づいて、成体マウスの脊髄からの培養ミクログリアにメソッドを記述します。この方法は、他の利用可能なプロトコルは14と比較して、文化の大人の細胞に迅速な方法を提供します。得られた製剤は70％であるミクログリア、残りのパーセンテージはアストロサイトから構成されている。私たちの文化の純度が他の公開された方法13に比べて低いが、この培養系は、主に脊髄に影響を与える病気の研究のために、強力なその中でだけでなく、様々な活性化刺激に文化応答にミクログリアを探索するのに便利です炎症反応は、主な機能です。 説明されているすべてのプロトコルはストーニーブルックの大学によって承認されているTY IACUC。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		 </tr>
		 <tr>
			<td>EDTA, 5mM</td>
			<td>Invitrogen</td>
			<td>15567-028</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Lidocaine, 60mM</td>
			<td>Sigma</td>
			<td>L-5647</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypan Blue</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypsin/EDTA</td>
			<td>Cellgro</td>
			<td>25-052-CI</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM, 1X</td>
			<td>Cellgro</td>
			<td>10-017</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium Pyruvate</td>
			<td>Cellgro</td>
			<td>25-000-Cl</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gentamycin Sulfate</td>
			<td>Biowittaker</td>
			<td>17-518Z</td>
			<td></td>
		 </tr>
		 <tr>
			<td>35 x 10mm tissue culture dish</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Poly-D-Lysine, 100 &mu;g/ml</td>
			<td></td>
			<td></td>
			<td>Dilute 1:20</td>
		 </tr>
		 <tr>
			<td>HBSS, 1X</td>
			<td>Cellgro</td>
			<td>20-023-CV</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

culturing microglia from the neonatal and adult central nervous system

ミクログリアのような、そのようなCNS開発の成熟、多発性硬化症、および脊髄損傷などの生理学的および病理学的プロセスにおいて非常に重要な役割を担っている、中枢神経系（CNS）の常駐マクロファージ様細胞であると。ミクログリアが活性化され、そのようなMSや脳卒中に起因する虚血性脳外傷で見られる軸索損傷などの神経損傷または刺激によって行動に動員することができます。 CNSこれらの免疫担当メンバーは、非病的条件下でのシナプス可塑性における役割を持つと考えられている。我々は、他のCNS細胞型および細胞培養デブリの存在のような交絡因子を最小にしながら生細胞数を最大にすることを目的としている新生児および成体組織から培養ミクログリアのためのプロトコルを採用している。我々は、全体のプロセスが実現可能となり再現大きく、容易に識別可能なCNSコンポーネント（ 例えば皮質脊髄セグメント）を利用する。成体細胞の使用多くの病状は主に出生後の脊髄に影響を与える研究としてsが、新生児の脳のミクログリアの使用に適した代替手段です。これらの培養系はまた、直接ミクログリア活性化を阻害または促進することができる化合物の効果を試験するために有用である。ミクログリアの活性化が成体CNS疾患の結果を形作ることができるので、新生児および成人ミクログリアを培養し検討することが可能なインビトロ系での必要性が存在する。

<html>安静時および活性化小グリア細胞の例を図1に示されている。ミクログリアは、めっき後24時間を可視化した（ 図1a）と分岐し（安静時）の形態を示した。プライミング試薬への暴露は、細胞のようなミクログリアの形態変化における細菌リポ多糖（LPS）の結果は、（図1b）がアクティブになります。 めっき用細胞を計数するの例...

Watch this Scientific Journal Video about Culturing Microglia from the Neonatal and Adult Central Nervous System at JoVE.com

Culturing Microglia from the Neonatal and Adult Central Nervous System

私たちは、新生児の大脳皮質および成体脊髄から実行可能なミクログリアの効率的かつ迅速に分離/培養のための方法を概説します。皮質ミクログリアの解剖とメッキが行われて、その後のミクログリアの収穫で、90分以内に達成することができます〜初期切開後10日。

新生児と大人の中枢神経系から培養ミクログリア

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

culturing-microglia-from-the-neonatal-and-adult-central-nervous-system

Research

JoVE Journal

Immunology and Infection

27.7K ビュー.  Stony Brook University. 私たちは、新生児の大脳皮質および成体脊髄から実行可能なミクログリアの効率的かつ迅速に分離/培養のための方法を概説します。皮質ミクログリアの解剖とメッキが行われて、その後のミクログリアの収穫で、90分以内に達成することができます〜初期切開後10日。

ビデオ: Culturing Microglia from the Neonatal and Adult Central Nervous System

Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1. These toxins are secreted into the extracellular milieu, but they act upon targets within the eukaryotic cytosol. Some AB toxins travel by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol2-4. In the ER, the catalytic A chain dissociates from the rest of the toxin and moves through a protein-conducting channel to reach its cytosolic target5. The translocated, cytosolic A chain is difficult to detect because toxin trafficking to the ER is an extremely inefficient process: most internalized toxin is routed to the lysosomes for degradation, so only a small fraction of surface-bound toxin reaches the Golgi apparatus and ER6-12.

To monitor toxin translocation from the ER to the cytosol in cultured cells, we combined a subcellular fractionation protocol with the highly sensitive detection method of surface plasmon resonance (SPR)13-15. The plasma membrane of toxin-treated cells is selectively permeabilized with digitonin, allowing collection of a cytosolic fraction which is subsequently perfused over an SPR sensor coated with an anti-toxin A chain antibody. The antibody-coated sensor can capture and detect pg/mL quantities of cytosolic toxin. With this protocol, it is possible to follow the kinetics of toxin entry into the cytosol and to characterize inhibitory effects on the translocation event. The concentration of cytosolic toxin can also be calculated from a standard curve generated with known quantities of A chain standards that have been perfused over the sensor. Our method represents a rapid, sensitive, and quantitative detection system that does not require radiolabeling or other modifications to the target toxin.

In this report, we describe how surface plasmon resonance is used to detect toxin entry into the host cytosol. This highly sensitive method can provide quantitative data on the amount of cytosolic toxin, and it can be applied to a range of toxins.

表面プラズモン共鳴によるホストのサイトゾルへ毒素の転座の検出

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1.  These toxins are secreted into the extracellular milieu, but they act upon ...

免疫・感染学

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

新生仔ラットの脳組織の混合グリア細胞培養物から主要なミクログリアの分離

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

ホット水 - フェノール抽出によるグラム陰性菌からの精製とリポ多糖の可視化

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

マウス新生児から保護免疫表現と機能皮質ミクログリアの分離

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

複製サイクルの初期段階を監視するためにB型肝炎ウイルスのレポーターシステムの開発

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

フローサイトメトリーによる中枢神経系からのミクログリアおよび単球由来マクロファージの解析

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

マウス小腸管免疫系からリンパ球を分離すること

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the central nervous system (CNS). Recent studies demonstrate the use of a peptide, RVG9R, incorporating the minimal receptor-binding domain of the Rabies virus glycoprotein, in eliciting the delivery of siRNA into neurons in the brain. In this protocol, the peptide-siRNA formulation is delivered intranasally with a pipette in the dominant hand, while the anesthetized mouse is restrained by the scruff with the nondominant hand in a "head down-and-forward position" to avoid drainage into the lung and stomach upon inhalation. This precise gripping of mice can be learned but is not easy and requires practice and skill to result in effective CNS uptake. Furthermore, the process is long-drawn, requiring about 45 min for the administration of a total volume of ~20-30 &#181;L of solution in 1-2 &#181;L droplet volumes per inhalation, with 3-4 min rest periods between each inhalation. The objective of this study is to disclose a mouse positioning device that enables the appropriate placement of mice for efficient IN administration of the peptide-siRNA formulation. Multiple features are incorporated into the design of the device, such as four or eight positioning chairs with adjustable height and tilt to restrain anesthetized mice in the head down-and-forward position, enabling easy visualization of the mice's nares and a built-in heating pad to maintain the mice's body temperatures during the procedure. Importantly, the ability to treat four or eight mice simultaneously with RVG9R-siRNA complexes in this manner enables studies on a much quicker time scale, for the testing of an IN therapeutic siRNA approach. In conclusion, this device allows for appropriate and controlled mouse head positioning for IN application of RVG9R-siRNA and other therapeutic molecules, such as nanoparticles or antibodies, for CNS delivery.

Here, we present a protocol using a mouse positioning device that enables the appropriate placement of mice for the intranasal administration of a brain-targeting peptide-siRNA formulation enabling effective gene silencing in the central nervous system.

中枢神経系への鼻腔内siRNA送達中におけるマウスの配置のための位置決め装置

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

実験的自己免疫性脳脊髄炎における中枢神経系におけるリンパ球浸潤のフロー細胞量分析

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier between the periphery and the CNS. The CNS is an immunologically specialized site, and in steady state conditions, immune privilege is most evident in the CNS parenchyma. In contrast, the meninges harbor a diverse array of resident cells, including innate and adaptive immune cells. During inflammatory conditions triggered by CNS injury, autoimmunity, infection, or even neurodegeneration, peripherally derived immune cells may enter the parenchyma and take up residence within the meninges. These cells are thought to perform both beneficial and detrimental actions during CNS disease pathogenesis. Despite this knowledge, the meninges are often overlooked when analyzing the CNS compartment, because conventional CNS tissue extraction methods omit the meningeal layers. This protocol presents two distinct methods for the rapid isolation of murine CNS tissues (i.e., brain, spinal cord, and meninges) that are suitable for downstream analysis via single-cell techniques, immunohistochemistry, and in situ hybridization methods. The described methods provide a comprehensive analysis of CNS tissues, ideal for assessing the phenotype, function, and localization of cells occupying the CNS compartment under homeostatic conditions and during disease pathogenesis.

This paper presents two optimized protocols for examining resident and peripherally derived immune cells within the central nervous system, including the brain, spinal cord, and meninges. Each of these protocols helps to ascertain the function and composition of the cells occupying these compartments under steady state and inflammatory conditions.

免疫細胞の下流分析のための中枢神経系組織および関連する髄膜の単離

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier ...