この作品は、研究助成金及び農業、林業、そして日本の水産省の食品ナノテクノロジープロジェクトからの補助金によって賄われていた。

1。肝臓の灌流<ol><li>ペントバルビタールナトリウム溶液（50 mgのナトリウムペントバルビタール/ kg体重）の腹腔内注射によって、成体雄Sprague - Dawleyラットを（体重150〜200グラム週齢の10〜15時）麻酔。 </li><li>ステンレス鍋に動物を配置し、腹部と胸部の上に70％エタノールをスプレー。 </li><li>皮膚の小さなカットして皮を作る。ハサミでカットで腹部を開きます。 </li><li>門脈の位置を確認し、門脈と疎塊の下に外科的スレッドを渡します。 </li><li>クランプ下大静脈と肝に血液の逆流を防ぐために、腸間膜静脈の遠位部分。はさみで横隔膜をカットして胸腔を開き、そして心を公開。 </li><li>門脈にプラスチック製のカテーテル（直径2mm）を挿入し、カテーテルの周囲にしっかりとスレッドを締めます。 </li><li> 37温めた℃、洗濯液（pH7.2のCa 2 +とMg 2 + 0.5mMのEGTA、10mMのHEPES、4.2 mMのNaHCO 3を含む無料のHBSSが）でロードされる灌流システムにカテーテルを接続する</li><li> 10ml /分の速度でその場で灌流を開始する。それと同時に、右心房の壁にカットを行います。 10分間の灌流を続ける。 </li><li>速度で20分- 、および10の浸み込ませる。[pH7.5の10mMのHEPESおよび4.2mM NaHCO 3を含むHBSSはI型コラゲナーゼ（0.05％）とトリプシン阻害剤（50μg/ ml）を補充した]コラゲナーゼ溶液への灌流液を切り替える10 ml /分、肝臓が少し腫れて漿膜下の肝臓の構造の崩壊のための符号を示すまで。 </li></ol> 2。実質の肝細胞が豊富な細胞分画の調製<ol><li>取り外し、慎重にコラゲナーゼ溶液の25mlを含む滅菌ビーカーに肝臓を移す。はさみで細かくミンチ。 </li><li>その結果、細胞懸濁液中に冷たいMEMの75 mlを加え。穏やかなピペッティングした後、ろ液は、セルストレーナー（100μm以下）を介して結合組織および未消化の組織片を除去する。 </li><li> 4℃（ブレーキオフ）で1分に50 × gで50 mlコニカルチューブと遠心分離機にろ液を移す。 </li><li>注意深く上清を捨てる。冷たいMEMでペレットを再懸濁します。 </li><li> ）三回2.4を繰り返します。 </li></ol> 3。肝細胞の混合初代培養<ol><li>に10％熱不活性化100μMのβ-メルカプトエタノールを添加したFCS、、10μg/ mlのインスリン、100μg/ mlのストレプトマイシンおよび100 U / mlペニシリンを含むDMEMで構成される成長培地に2.5で得られた細胞）を一時停止、および種子6.7 × 10 4細胞/ cm 2（培地の5 × 10 6細胞/フラスコ/ 12-15 ml）の密度で：五から十組織培養フラスコ（75 cm 2と表面積）。 </li><li>文化のflasksat 37インキュベート℃、5％CO 2の大気中のC - 95％の空気。 </li><li>増殖培地2〜3日おきの間隔を交換してください。 </li></ol> 4。マクロファージの選択的分離<ol><li>文化の12日後に、マクロファージは、細胞シートで増殖する。これらの細胞は80℃培養フラスコの揺れ逆数で中断している - 30分間毎分120ストロークを37℃ </li><li> 100ミリメートルの非組織培養グレードのプラスチック製の皿に培養液を移す。 1つのプラスチックの皿に2つのT75フラスコから培地をプール。成長培地を補充培養フラスコをインキュベーターに戻す。 </li><li> 37℃で30分間プラスチック皿をインキュベート℃、5％CO 2の大気中のC - 95％の空気。他の混入細胞はそうではないマクロファージが容易に、このインキュベーションのプロセスの下で、非組織培養グレードのプラスチックの皿に取り付けます。 </li><li>培地を吸引し、穏やかにPBSでプラスチック製の皿を洗う。このステップを3回繰り返します。 </li><li>皿にTrypLE Expressソリューションの1 mlを加え、そして37℃で10分間インキュベート℃、5％CO 2の大気中のC - 95％の空気。 </li><li>増殖培地9mlのを追加。優しくセルスクレイパーで接続されているマクロファージを削り取る。 </li><li> 25 ° C（上ブレーキ）で5分で180 × gで15 mlコニカルチューブと遠心分離機に細胞懸濁液を移す。 </li><li>上清を捨て、増殖培地1 mlを加える。活発なピペッティングによって単一細胞に解離する。血球計計数することにより、細胞数を列挙する。 </li><li>分離ステップは、二週間以上の2〜3日間隔で同一の培養フラスコで繰り返すことができます。 </li><li>分離されたマクロファージは、3.1で説明されている増殖培地で培養することができる。 </li></ol> 5。代表的な結果成体ラット肝細胞の混合初代培養の例を図2に示されています。肝実質細胞は培養中の数日以内に上皮細胞の形態を失い、変性または線維芽細胞様細胞に変身。その後、一日中6〜12段階、コントラスト、明るい、ラウンドマクロファージ様細胞はvigourouly線維芽細胞シートで増殖する。培養フラスコを振とうすることにより、マクロファージは、容易に培養培地に懸濁させ、そしてマクロファージの選択的接着（図3）のプラスチック製の皿の結果でその後の転送とインキュベーション。マクロファージ様細胞は、8日目には早くも収穫され、数字は12〜14日に最大レベルに到達することができます。以上の10 6の細胞をT75培養フラスコあたり10 7（図3）のフラスコあたりの総細胞収率を可能にする、以上の二週間に2〜3日間隔で繰り返しT75培養フラスコから収穫することができる。分離されたマクロファージの純度は次のようなED - 1（図4）、ED - 3、OX - 41（図4）とIba1 9などのラットマクロファージ特異的抗体、とフローサイトメトリーまたは免疫細胞化学により評価、95から99パーセントでした。分離した細胞は、ポリスチレンビーズのアクティブな食作用（図5）、組換えGM - CSF、LPSで刺激し、多核巨細胞9の形成時に炎症/抗炎症性サイトカインの分泌に対する増殖応答としてマクロファージの典型的な特徴を、示す。 <img src="/files/ftp_upload/2757/2757fig1.jpg" alt="図1" /> 図1ラット肝細胞の混合初代培養からのマクロファージ様細胞の選択的単離および精製の ​​ためのスキーム。 <img src="/files/ftp_upload/2757/2757fig2.jpg" alt="図2" /> 図2。ラット初代肝細胞の培養とマクロファージ様細胞の増殖。矢印は多核巨細胞を示している。スケールバー= 100μmの。エルゼビアから許可を得て免疫学的方法の研究、Vol.360、47〜55（2010）9から転載。 <img src="/files/ftp_upload/2757/2757fig3.jpg" alt="図3" /> 図3。揺れや添付法によるマクロファージ様細胞の選択的分離。細胞は、フラスコを振とうすることにより培養液に懸濁し、その後、非組織培養グレードのプラスチック製の皿に移し、37℃でインキュベートした。他の混入繊維芽細胞が中断されたままながら、めっき後の10分には早くも、マクロファージ様細胞は、皿の表面に付着。 PBSで洗浄した後、高度に精製されたマクロファージの人口が得られた。これらの細胞は、培養の40分後に典型的なマクロファージの形態を獲得し、有糸分裂細胞は、頻繁に（矢印）が観察された。異なる培養期間におけるフラスコから回収した細胞数に変更があった場合、それが表示されます。値は3〜5フラスコに、平均± SDである。実験を3回繰り返していたし、データは別の記号で示されます。スケールバー= 100μmの。エルゼビアから許可を得て免疫学的方法の研究、Vol.360、47〜55（2010）9から転載。 <img src="/files/ftp_upload/2757/2757fig4.jpg" alt="図4" /> 図4ラットマクロファージに対するモノクローナル抗体を用いたマクロファージ様細胞の免疫染色。 <img src="/files/ftp_upload/2757/2757fig5.jpg" alt="図5" /> 図5。マクロファージ様細胞によるFITC標識マイクロビーズの貪食。

<ol>
	<li>Crispe, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+liver+as+a+lymphoid+organ.">The liver as a lymphoid organ.</a> Annu. Rev. Immunol. 27, 147-163 (2009).</li><li>Roberts, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+Kupffer+cell+in+mediating+hepatic+toxicity+and+carcinogenesis.">Role of the Kupffer cell in mediating hepatic toxicity and carcinogenesis.</a> Toxicol. Sci. 96, 2-15 (2007).</li><li>Gao, B., Jeong, W. I., Tian, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver:+An+organ+with+predominant+innate+immunity.">Liver: An organ with predominant innate immunity.</a> Hepatology. 47, 729-736 (2008).</li><li>Janousek, J., Strmen, E., Gervais, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+murine+Kupffer+cells+by+centrifugal+elutriation.">Purification of murine Kupffer cells by centrifugal elutriation.</a> J. Immunol. Methods. 164, 109-117 (1993).</li><li>Olynyk, J. K., Clarke, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+primary+culture+of+rat+Kupffer+cells.">Isolation and primary culture of rat Kupffer cells.</a> J. Gastroenterol. Hepatol. 13, 842-845 (1998).</li><li>Heuff, G., Meyer, S., Beelen, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+rat+and+human+Kupffer+cells+by+a+modified+enzymatic+assay.">Isolation of rat and human Kupffer cells by a modified enzymatic assay.</a> J. Immunol. Methods. 174, 61-65 (1994).</li><li>Seglen, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+isolated+rat+liver+cells.">Preparation of isolated rat liver cells.</a> Methods Cell Biol. 13, 29-83 (1976).</li><li>Yoshioka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+and+expression+of+cytokine+mRNAs+by+lipopolysaccharide+in+bovine+Kupffer+cells.">Primary culture and expression of cytokine mRNAs by lipopolysaccharide in bovine Kupffer cells.</a> Vet. Immunol. Immunopathol. 58, 155-163 (1997).</li><li>Kitani, H., Takenouchi, T., Sato, M., Yoshioka, M., Yamanaka, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+isolation+method+for+macrophage-like+cells+from+mixed+primary+cultures+of+adult+rat+liver+cells.">A novel isolation method for macrophage-like cells from mixed primary cultures of adult rat liver cells.</a> J. Immunol. Methods. 360, 47-55 (2010).</li><li>Gorrell, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+fibrosis:+the+hepatocyte+revisited.">Liver fibrosis: the hepatocyte revisited.</a> Hepatology. 46, 1659-1661 (2007).</li></ol>

すべての動物は、動物実験のための機関の委員会が定めたガイドラインと規則に従って、動物衛生研究所の動物施設に保持され、運営された。

ここで、我々は、成体ラットの肝細胞の混合一次培養物からマクロファージを得るために簡単かつ効率的な方法を報告する。私たちの方法は、マクロファージ9として、その生物学的特性に基づいてこれらの細胞のその後の単離および精製してラットの肝細胞の混合培養におけるマクロファージの小説増殖活性に最初に依存しており、。それは、大人の肝臓の細胞の混合初代培養で増殖したマクロファージはクッパーまたは肝実質細胞の画分に汚染された関連する細胞由来のかもしれない可能性があります。マクロファージが混在初代培養における実質肝細胞10と他の繊維芽細胞の形質転換に起因する特定の環境の変化に対応している場合があります。 結論として、私たちの方法は、高度な機器や技術的なスキルを用いてラット肝臓cellswithoutの初代培養から十分な数と純度のマクロファージ様細胞の分離を提供します。さらに、分離手順は、2週間以上同じ培養フラスコで繰り返すことができます。このメソッドは、他の哺乳動物種に適用できるかもしれない。

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td>ペントバルビタールナトリウムソリューション</td><td>共立製薬</td><td> Somnopentylインジェクション</td><td></td></tr><tr><td>ハンクス平衡塩溶液（HBSS） </td><td>インビトロジェン</td><td> 14185-052 </td><td></td></tr><tr><td>エチレングリコール四酢酸（EGTA） </td><td>シグマアルドリッチ</td><td> E - 4378 </td><td></td></tr><tr><td>コラゲナーゼ</td><td>和光純薬</td><td> 032から10534 </td><td>細胞解離グレード （ロットチェックに必要） </td></tr><tr><td>トリプシン阻害剤</td><td>シグマアルドリッチ</td><td> T - 9128 </td><td></td></tr><tr><td>イーグルの最小必須培地（MEM） </td><td>シグマアルドリッチ</td><td> M - 4655 </td><td></td></tr><tr><td>ダルベッコは変更されている イーグル培地（DMEM） </td><td>シグマアルドリッチ</td><td> D - 6459 </td><td>高グルコース型</td></tr><tr><td>ウシ胎児血清（FCS） </td><td> HyClone </td><td> SH30070.03 </td><td> 56で不活性化熱℃で30分間。 （ロットチェックに必要） </td></tr><tr><td> TrypLEエクスプレス</td><td>インビトロジェン</td><td> 12605 </td><td></td></tr><tr><td>ダルベッコのリン酸緩衝生理食塩水（PBS） </td><td>インビトロジェン</td><td> 14190 </td><td>のCa 2 +とMg 2 +フリー</td></tr><tr><td> β-メルカプトエタノール</td><td>シグマアルドリッチ</td><td> M - 7522 </td><td>在庫：蒸留水に100mMの</td></tr><tr><td>インスリン</td><td>シグマアルドリッチ</td><td> I - 5500 </td><td>在庫：0.1N塩酸で10 mg / mlの</td></tr><tr><td>ペニシリン/ストレプトマイシン</td><td>インビトロジェン</td><td> 15070 </td><td></td></tr><tr><td>セルストレーナー</td><td> BDバイオサイエンス</td><td> 352360 </td><td>メッシュサイズ：100μm以下</td></tr><tr><td>組織培養フラスコ</td><td>住友ベークライト株式会社</td><td> MS - 21250 </td><td>表面積：75 cm 2と </td></tr><tr><td>非組織培養プラスチック皿</td><td> BDバイオサイエンス</td><td> 351005 </td><td></td></tr><tr><td>セルスクレイパー</td><td>コー​​ニング（株） </td><td> 3010 </td><td></td></tr><tr><td>逆数シェーカー</td><td>タイテック</td><td> NR - 1 </td><td></td></tr></tbody></table>

a simple and efficient method to isolate macrophages from mixed primary cultures of adult liver cells

クッパー細胞は肝臓特異常駐マクロファージであると1-3肝臓の生理学的および病理学的機能において重要な役割を果たしている。肝臓のマクロファージの単離方法は4-6をよく説明されてきたが、これらのメソッドのほとんどは、遠心水簸機や技能などの高度な機器を、必要とする。模式的に図1に示すように、ここで、我々は、成体ラットの肝細胞の混合一次培養物から十分な数と純度で肝臓のマクロファージを取得するための新しい方法を提供する。 二段階灌流法7,8により肝細胞の解離した後、主に肝実質細胞で構成される画分を調製し、DMEM、10％FCS.Parenchymal肝細胞で構成される培地とT75組織培養フラスコに播種されると、上皮細胞の形態を失う文化の中で数日以内に、変性または線維芽細胞様細胞（図2）に変換する。位相コントラスト - 明るい6日目の周りの文化が進む、、として、ラウンドマクロファージ様細胞は、線維芽細胞シート（図2）で増殖し始める。マクロファージ様細胞の増殖は、フラスコの表面上に細胞シートをカバーする、継続すると一日約12最大レベルに達する。培養フラスコを振とうすることにより、マクロファージは、容易に培養培地中に懸濁させる。のような他の汚染細胞が中断されたままマクロファージ（図3）、の選択的接着のプラスチック製の皿の結果でその後の転送と短いインキュベーション。 PBSでいくつかのリンスした後、添付されたマクロファージが収穫されます。 10以上の6個の細胞が二週間以上（図3）をそれぞれ2〜3日間隔で同じT75組織培養フラスコから繰り返し収穫することができる。フローサイトメトリーにより評価として孤立マクロファージの純度は、95〜99パーセントだったかラットマクロファージ特異的抗体（図4）と免疫細胞化学は。分離した細胞は、LPSで刺激、との形成時にポリスチレンビーズのアクティブな食作用（図5）、組換えGM - CSFに対する増殖応答、​​炎症/抗炎症性サイトカインの分泌を示す多核巨細胞9。 結論として、我々は複雑な装置なしで十分な数と純度で肝臓のマクロファージを得るために簡単かつ効率的な方法を提供し、skills.This方法は、他の哺乳動物種に適用できるかもしれない。

Watch this Scientific Journal Video about A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells at JoVE.com

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

ラット肝細胞の初代培養からマクロファージを取得するための新しい方法が記載されている。このメソッドは、プラスチック製の皿に選択的付着によって培養フラスコ及び精製の揺れが続く文化におけるマクロファージの増殖を、利用しています。この手法は、効率的に複雑な装置やスキルがなくても肝臓のマクロファージを提供します。

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 ...

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Immunology and Infection

37.9K ビュー.  National Institute of Agrobiological Sciences, Tsukuba, Japan. ラット肝細胞の初代培養からマクロファージを取得するための新しい方法が記載されている。このメソッドは、プラスチック製の皿に選択的付着によって培養フラスコ及び精製の揺れが続く文化におけるマクロファージの増殖を、利用しています。この手法は、効率的に複雑な装置やスキルがなくても肝臓のマクロファージを提供します。

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

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 ...

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 ...

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-&kappa;B and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

フローサイトメトリーによるヒト好中球の核因子の活性を検出するために簡単で効率的な方法

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

ヌクレオによるヒトTHP-1マクロファージの高効率トランスフェクション

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

マウスの樹状細胞サブセットの単離のための効率的かつ高収率方法

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

突然変異体ワクシニアウイルスベクターを構築するためのシンプルで効率的なアプローチ

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

マウスの骨髄からGM-CSF由来肺胞のようなマクロファージや樹状細胞の生成および同定

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

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 ...

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

結膜から細胞を単離して表現する非侵襲的方法

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

NK と慢性的なアクティブなエプスタイン ‐ バーウイルス感染症から T 細胞株を確立する効率的かつ簡単な方法

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...