制服サービス大学の学内プログラムによって資金を供給。

1。新生仔ラットの脳組織の解剖<ol><li>チルリーボビッツのL-15培地（レイボビッツL-15 + 0.1％BSA + 1％のペン/連鎖球菌）に4℃暖かい培地（DMEM + 10％FBS + 1％ペニシリン/ストレプトマイシン）37℃氷の上でエアコンL-15メディアの4-5 mlの4 60X15ミリメートルのペトリ皿を準備します。 100％エタノールを持つすべての手術器具を滅菌する。 </li><li> 70％エタノールでP2新生児ラップ子犬をすすぎます。 （P1-P5歳新生児仔ラットは、このプロトコルで使用することができます。） </li><li>迅速に滅菌鋭いハサミで子犬ラットを刎ねると、70％エタノールにすぐに頭をドロップします。 5仔ラットの合計繰り返した後食塩水に頭を転送します。 </li><li>氷上で5ミリリットルL-15溶液（レイボビッツL-15 + 0.1％BSA + 1％ペン/連鎖球菌） - 4のペトリ皿に頭と場所から全体の頭脳を削除します。 </li><li> 1.4リットルの残りの子犬のために - 手順1.2を繰り返します。 </li><li>脳から髄膜を除去し、DESを転送氷の上でL-15溶液（レイボビッツL-15 + 0.1％BSA + 1％のペン/連鎖球菌）の4-5 mlの新しいペトリ皿に赤外LEDの脳組織（ すなわち 、大脳皮質、小脳、全脳、など）。 </li></ol> 2。混合グリア細胞集団の準備<ol><li>ハサミや刃で脳組織を損傷することなく、滅菌50 mlコニカルチューブに10 mlのピ​​ペットで組織を転送します。 </li><li> 4℃で5分間2500 RCFで円錐形の遠心</li><li>上清を吸引除去する。新鮮なL-15培地（レイボビッツL-15 + 0.1％BSA + 1％のペン/連鎖球菌）の4-5 mlを追加します。 </li><li>滅菌した10 mlのピ​​ペットでピペットは、組織の上下に10回。 </li><li>新鮮な50 mlコニカルチューブにセルストレーナー（100μm以下の細孔）を配置します。上下にピペット組織は一度滅菌5mlのピペットを用いて、セルのストレーナーにフラッシュピペットを用いて、コニカルチューブに細胞ストレーナーを通して材料を分配する。 </li><li>新鮮なL-15培地（レイボビッツL-15 + 0.1％BS 4-5 mlのセルストレーナーを洗浄します+ 1％のペン/連鎖球菌）。 </li><li> 4℃で5分間で2,500 RCFで緊張した細胞と円錐形の遠心分離</li></ol> 3。混合グリア細胞培養のメッキとメンテナンス<ol><li>処理された各ラット子犬の脳については、各フラスコに培養培地（DMEM + 10％FBS + 1％ペニシリン/ストレプトマイシン）の12 mlを加えて1滅菌T-75フラスコを準備します。 </li><li>ペレット化した細胞からの上清を吸引除去し、細胞ペレットに培地の5-6 mlを加える。 10 mlのピ​​ペットで10回ピペッティングします。 </li><li>それぞれのT-75フラスコに細胞懸濁液の等量を転送します。 </li><li> 1-3週間の合計で37℃5％CO 2インキュベーター内でフラスコをインキュベートします。 </li><li>フラスコは、最初の5日間インキュベートし、5日目に、新鮮な培地12 mlを各フラスコに培地を交換し、インキュベーターに戻す必要があります。その後、3日ごとに、合流を達成するために新鮮な培地を12 mlの各フラスコ内の培地を交換してください。これは何されている必要があり細胞が付着フラスコの底に触れることなく、非常に慎重にね。 </li></ol> 4。プライマリミクログリアの分離とめっき<ol><li>混合グリア培養が完全にコンフルエントになったら、インキュベーターからフラスコを削除し、環境大気とのガス交換を防ぐためパラフィルムでフラスコのキャップをカバーしています。 </li><li> 37℃で1時間に100 RPS（ラボコンパニオンSI-600、Jeioテック）でフラスコを振る℃に</li><li>フラスコ表面に星状膠細胞層を中断せずに10 mlのピ​​ペットを用いてすべてのフラスコから培地を収集します。 50 mlコニカルチューブにメディアを配置します。インキュベーターにフラスコと戻りフラスコに新鮮な培地を追加します。 </li><li> 4℃で5分間で2,500 RCFでコニカルチューブを遠心分離</li><li>すべてのコニカルチューブから上清を吸引除去する。細胞ペレットは、高純度のミクログリア細胞である。ミクログリアメッキ培地（DMEM + 10％FES + 1％ペニシリン/ストレプトマイシン）を1mlに再懸濁したペレット。 </li><li>を使用して再懸濁した培地で細胞密度を数える血球。 </li><li>ミリリットル当たりの密度2×10e5細胞を達成するために、ミクログリアメッキメディアの適切なボリュームを追加します。実験的な解析に適したプレート。 </li><li>ミクログリアは一晩にアタッチすることができます。 </li></ol> 5。代表的な結果高純度の主ミクログリア培養の結果、上記のプロトコル。としてマクロファージ/ミクログリア細胞特異的マーカー（Iba1）の免疫組織化学によって決定される、メッキミクログリア培養は&gt; 90％純粋（ 図2）です。さらに、アストロサイト、オリゴデンドロサイトとニューロン細胞特異的マーカーを染色すると、最小限の汚染（ 図2）を示しています。混合グリア細胞培養を確立した後、ミクログリアは4つの連続した​​回まで振とうすることにより単離することができる。初期ミクログリアの分離は、約2.2×106細胞/ mlまたは10フラスコ当たり約9万個の細胞が得られると予想される収量は、それぞれの連続した​​SHAとともに減少するKE。この方法により単離されたミクログリアが貪食能力など、一酸化窒素の生産として機能し、神経毒性（ 図3）13 から14を調査するために首尾よく使用されています。 <img alt="図1" src="/files/ftp_upload/3814/3814fig1.jpg" /> 図1混合グリア細胞培養、プライマリミクログリアの分離を準備するためのプロトコルの概要。 <img alt="図2" src="/files/ftp_upload/3814/3814fig2.jpg" /> ミクログリア（IBA-1、赤）、アストロサイト（GFAP、緑）、ニューロン（NeuN、赤）とオリゴデンドロサイト（CC1、赤）は、特定のマーカーを用いて免疫組織化学的分析によって検証として高純度のミクログリアの図2。分離。 DAPI（青）で培養中の細胞のDNAを染色も表示されます。 <img alt="図3" src="/files/ftp_upload/3814/3814fig3.jpg" /> 図3ミクログリアは、メソッドdescriで分離されたベッドは、貪食アッセイ、機能アッセイと神経毒性の研究に限定を含むではなく、多くのアッセイで使用されています。これらの研究で、我々が制御するために露出したミクログリア（IBA-1陽性、緑）、（）またはLPS（B）が扱われているメディアを発見した、定量することができる蛍光標識ラテックスビーズ（赤）、それらの貪食を増加させる（ C）。さらに、LPS（1ng/ml）治療は、ミクログリア（D）で一酸化窒素の産生を増加させることができます。最後に、トランスウェルインサート区切りまたは直接ミクログリア - ニューロン培養の両方を介して、我々は、乳酸脱水素酵素（LDH）放出（E、F）によって測定されたLPSとともにインキュベートしたミクログリアが神経細胞死を誘導できることを見出した。

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利害の衝突が宣言されません。

このプロトコルは定期的に研究実験のための純粋で健康的なミクログリアを製造するために利用されていますが、手順の重要な部分の間に技術的な側面を慎重に検討は、単離されたミクログリアの変動を最小限に抑えることができます。まず、タイムリーで働く新生児の仔ラットの脳組織の解剖中に組織への酸素および虚血性損傷を最小限にする必要があります。しかし、髄膜の存在がこれ?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> 60ミリメートルX 15ミリメートルのペトリ皿</td><td>フィッシャーブランド</td><td> 0875713A </td><td></td></tr><tr><td>シャープ解剖はさみ</td><td>ファイン科学ツール</td><td> 14094から11 </td><td></td></tr><tr><td>デュモン＃曲線7bの鉗子標準のヒント、11センチメートル</td><td>ファイン科学ツール</td><td> 11270から20 </td><td></td></tr><tr><td>デュモン＃5鉗子標準のヒント、ストレート、11センチメートル</td><td>ファイン科学ツール</td><td> 11251から10 </td><td></td></tr><tr><td> 50 mlコニカル遠心チューブ</td><td> VWR </td><td> 89039-656 </td><td></td></tr><tr><td> 5ミリリットル血清ピペット</td><td>グルニエバイオOne </td><td> 606180 </td><td></td></tr><tr><td> 10ミリリットル血清ピペット</td><td>グルニエバイオOne </td><td> 607180 </td><td></td></tr><tr><td> 100μmの滅菌ナイロンセルストレーナー</td><td>ファルコン</td><td> 35から2360 </td><td></td></tr><tr><td> 75センチメートル2組織培養フラスコ</td><td>コー​​ニング</td><td> 430641 </td><td></td></tr><tr><td>ダルベッコの最小必須培地（DMEM） </td><td> GIBCO（インビトロジェン社） </td><td> 31053-028 </td><td></td></tr><tr><td>リーボビッツのL-15培地</td><td> GIBCO（インビトロジェン社） </td><td> 11415064 </td><td></td></tr><tr><td>ウシ胎仔血清</td><td> GIBCO（インビトロジェン社） </td><td> 16000-036 </td><td></td></tr><tr><td>胎児のウマ血清</td><td>フィッシャー</td><td> SH3007402 </td><td></td></tr><tr><td>ペニシリン - ストレプトマイシン</td><td> GIBCO（インビトロジェン社） </td><td> 15140163 </td><td></td></tr><tr><td> 100％エタノール</td><td>ワーナー·グラハム·カンパニー</td><td> 64-17-5 </td><td></td></tr><tr><td>リン酸塩は、食塩水、10X、pH7.4を緩衝</td><td>質生物学、株式会社。 </td><td> 119-069-131 </td><td>無菌の蒸留水で1X </td></tr><tr><td>バイオハザードバッグ</td><td> VWR </td><td> 14220-028 </td><td></td></tr><tr><td> Haemocytometer </td><td> Hausser科学</td><td> 1492 </td><td></td></tr><tr><td> cellBIND表面を有する6ウェル細胞培養プレート</td><td>コー​​ニング</td><td> 3335 </td><td></td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue at JoVE.com

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

脳の細胞の不均質性からプライマリミクログリアを分離すると、生理学的および病理学的条件の両方で彼らの役割を調査することが不可欠である。このプロトコルは、機械的な分離と高収率かつ高純度を提供する混合細胞培養技術、実行可能な主なミクログリア細胞について説明します。<em&gt; in vitroで</em&gt;研究と下流のアプリケーションに最適です。

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 ...

primary-microglia-isolation-from-mixed-glial-cell-cultures-neonatal

Research

JoVE Journal

Immunology and Infection

39.1K ビュー.  Uniformed Services University. 脳の細胞の不均質性からプライマリミクログリアを分離すると、生理学的および病理学的条件の両方で彼らの役割を調査することが不可欠である。このプロトコルは、機械的な分離と高収率かつ高純度を提供する混合細胞培養技術、実行可能な主なミクログリア細胞について説明します。 in vitroで研究と下流のアプリケーションに最適です。

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

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

成人肝細胞の混合初代培養からマクロファージを分離するためにシンプルかつ効率的な方法

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

免疫・感染学

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

初代ヒトケラチノサイトからカドヘリン文化の生成

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

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 ...

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 ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

単離とマウス骨髄由来の単球の静脈注射

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

ラット精巣からのセルトリ細胞と尿細管周囲細胞の単離

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

虚血性マウス脳からの免疫細胞の単離およびフローサイトメトリー分析

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

マウスからの初代肝細胞培養における炎症反応の誘導

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression of proteins known to respond to metabolic stress was measured in brain-nuclei isolates using Western blotting. This was done to assess whether metabolic stress-induced nutrient insufficiency in the body triggered neuronal stress. We have previously demonstrated that nutrient insufficiency in neonates elicits metabolic stress in the gut. Furthermore, colostrum oxytocin modulates cellular stress response, inflammation, and autophagy markers in newborn rat gut villi prior to and after first feed. Signaling protein markers associated with the endoplasmic reticulum stress [ER chaperone binding immunoglobulin protein (BiP), eukaryotic translation initiation factor 2A (eIF2a), and eIF2a kinase protein kinase R (p-PKR)], as well as two inflammation-signaling proteins [nuclear factor-&#954;B (NF-kB) and inhibitor &#954;B (IkB)], were measured in newborn brain nuclei [nucleus of the solitary tract (NTS), paraventricular nucleus (PVN), supra-optic nucleus (SON), cortex (CX), striatum nuclei (STR), and medial preoptic nucleus (MPO)] before the first feed (unprimed by colostrum) and after the start of nursing (primed by colostrum). Expression of BiP/GRP78 and p-eIF2a were upregulated in unprimed and downregulated in primed NTS tissue. NF-kB was retained (high) in the CX, STR, and MPO cytoplasm, whereas NF-kB was lower and unchanged in NTS, PVN, and SON in both conditions. The collective BiP and p-eIF2 findings are consistent with a stress response. eIf2a was phosphorylated by dsRNA dependent kinase (p-PKR) in the SON, CX, STR, and MPO. However, in the NTS (and to a lesser extent in PVN), eIf2a was phosphorylated by another kinase, general control nonderepressible-2 kinase (GCN2). The stress-modulating mechanisms previously observed in newborn gut enterocytes appear to be mirrored in some OTR-rich brain regions. The NTS and PVN may utilize a different phosphorylation mechanism (under nutrient deficiency) from other regions and be refractory to the impact of nutrient insufficiency. Collectively, this data suggests that brain responses to nutrient insufficiency stress are offset by signaling from colostrum-primed enterocytes.

Here, we present a protocol to isolate brain nuclei in the neonatal rat brain in conjunction with first colostrum feeding. This technique allows the study of nutrient insufficiency stress in the brain as modulated by enterocyte signaling.

前に、最初の初乳の授乳後細胞ストレスと新生児ラットにおける離散のオキシトシン分泌脳核の炎症を評価します。

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression ...

Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central nervous system (CNS). The increasing evidences of their involvement in the control of infectious diseases justify the emerging interest in the improvement of methodologies to isolate primary astrocytes and microglia in order to evaluate their responses to infections that affect the CNS. Considering the impact of Trypanosoma cruzi (T. cruzi) and Toxoplasma gondii (T. gondii) infection in the CNS, here we provide a method to extract, maintain, dissociate and infect murine astrocytes and microglia cells with protozoa parasites. Extracted cells from newborn cortices are maintained in vitro for 14 days with periodic differential media replacement. Astrocytes and microglia are obtained from the same extraction protocol by mechanical dissociation. After phenotyping by flow cytometry, cells are infected with protozoa parasites. The infection rate is determined by fluorescence microscopy at different time points, thus enabling the evaluation of differential ability of glial cells to control protozoan invasion and replication. These techniques represent simple, cheap and efficient methods to study the responses of astrocytes and microglia to infections, opening the field for further neuroimmunology analysis.

The overall goal of this protocol is to instruct how to extract, maintain, and dissociate murine astrocyte and microglia cells from the central nervous system, followed by infection with protozoa parasites.

原虫性原虫感染に対する原発星およびミクログリアの同時分離

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central ...