This work was supported by grants from CONACyT-SEP (SEP-2008-84582; CB-2011-01-168497) and Promep-SEP for R.A.G.; P.H. received a scholarship from CONACyT (447442).

1. HFF細胞培養およびAd-感染<ol><li>以前に説明したように8 HFF細胞上の蛍光形成単位（FFU）として、HEK-293細胞と力価の単層中のAd5のWTウイルスを伝播します。 </li><li>無菌培養で37ºCで100mmの皿および5％CO 2加湿インキュベーター中DMEM / 10％ウシ胎児血清（FBS）10ml中のヒト包皮線維芽細胞（HFF）を成長させます。 4つの16平方セットで細胞を計数することによってノイバウアーチャンバーを用いて細胞数を決定します。 1ml当たりの細胞数は、4つの16平方セットにおける細胞の平均数を計算し、10 4が 、この数を乗じて得られる。ステップ1.3に含まれる各時間後の感染のために、1×10 7個のHFF細胞を用います。 </li><li> 30フォーカス形成単位、またはFFUのMOIで皿当たりDMEM中でのAd5の1ミリリットルを使用して、100mmの組織培養皿にアデノウイルス5型（Ad5）野生型（WT）（H5pg4100 8）とHFF細胞をモック感染または感染、セルあたり。ああで2時間インキュベートします慎重に細胞の上にウイルス接種物の均一な分布を確実にするために15分毎に料理をロッキングumidified細胞培養37ºCでインキュベータ、5％CO 2、。この時間の後、培地を除去し、10％FBSを補充した新鮮なDMEMを加え、16、24または36º37℃の加湿細胞培養インキュベーター中で時間、5％CO 2インキュベートします。 2.1に進みます。 </li></ol>アデノウイルスRCで強化されたサブ核画分の調製<ol><li>収穫のAd5感染または細胞スクレーパーでモック感染したHFF細胞および滅菌遠心管で細胞を集めます。ステップ1.2のように細胞数を決定します。ステップ1.3で指定された各時間後の感染のために、1×10 7個の細胞を使用してください。 </li><li> 220×gで、5分間、4ºCで細胞を遠心します。 </li><li>氷冷PBS（137mMの塩化ナトリウム、2.7のKCl、10mMのの Na 2 HPO 4、1.8mMのKH 2 PO 4）で細胞ペレットを再懸濁します。洗浄細胞ペレット1回の洗浄につき氷冷PBS 5mlのを使用して3回。この目的のために、220×gで遠心分離細胞は、5分間の4ºC、デカントして捨て、上清（SN）を、穏やかなピペッティングにより細胞ペレットを再懸濁します。 </li><li>プラズマ膜を破壊するために、氷冷低張緩衝液（10mMのHEPES pHは7.9、10mMの塩化カリウム、1.5mMのMgCl 2を、0.5 mMのDTT、20μG / mlのフェニルメチルスルホニルフルオリド（PMSF）の700μLに細胞ペレットを再懸濁、10μグラム/ mlのウシ膵臓トリプシン阻害剤、10μグラム/ mlのペプスタチンA、10μグラム/ mlのN -アセチル-L-ロイシル-L-ロイシル- Lを含むシステイン、セリン、スレオニン及びアスパラギン酸プロテアーゼ阻害剤の混合物-argininal）、細胞を3時間氷上で膨潤させます。 </li><li>溶解ゆったり型テフロン乳棒でホモジナイザーを用いて細胞。全ての細胞が溶解されていることを確認するために、明視野顕微鏡で80ダウンスストロークと監視のサンプルごとに20ストロークを実行しますが、核は、nを持っていることOT破損または破裂して。 </li><li> 300×gで、5分間、4ºCで細胞ホモジネートを遠心します。遠心管で-20ºCでの細胞質画分としてSN保管してください。 </li><li>核から細胞破片を除去するために、（0.25 Mスクロース、10mMのMgCl 2を20μG / mlのPMSF、システインの混合物、セリン、10を含むスレオニンおよびアスパルチルプロテアーゼ阻害剤液1（S1）の750μLにペレットを再懸濁μG / mlのウシ膵臓トリプシンインヒビター、10μG / mlのペプスタチンA、10μG / mlのN -アセチル- L -ロイシル-L-ロイシル-L- argininal）と溶液2等量の上の層（S2） （0.35 Mスクロース、0.5mMのMgCl 2を、20μグラム/ mlのPMSF、10μグラム/ mlのウシ膵臓トリプシンインヒビターを含む、システイン、セリン、スレオニン及びアスパラギン酸プロテアーゼ阻害剤の混合物、10μグラム/ mlのペプスタチンA、10μグラム/ mlのN -アセチル-L-ロイシル-L-ロイシル-L-argininal）。 CENTR1400×gで、5分間、4ºCでifuge。 </li><li>マイクロピペットを使用してSNを破棄します。ペレットを乱さないようにしてください。 </li><li> S2の750μLで孤立した核を含むペレットを再懸濁します。 </li><li>アデノウイルスRC（RCF）で強化された核内画分を単離するために、2つの5分のパルスを用いて、超音波浴で核を再懸濁し超音波処理、または明視野顕微鏡により観察されるように、すべての核が溶解するまで。 Cで、または4°未満のサンプルを維持するために必要に応じて超音波浴中に氷を使用​​してください。 </li><li>溶液3（S3）3000×gで（0.88 Mスクロース、0.5のMgCl 2）、遠心分離、10分間4ºC等量の上で超音波処理した核レイヤ。 -70ºCで核質画分（NPL）と1.5ミリリットルの上清を保管してください。 -70ºCでRCFとしてS2の700μLでペレットや店舗を再懸濁します。 </li></ol> RCFの3ウエスタンブロット分析注：NPLとRCF画分sのウエスタンブロット分析のためにステップ2.11で得られた総容量とは別にらNPLのための640μLとRCFのための300μリットル。 <ol><li> Laemmli緩衝液の2倍（4％SDS、20％グリセロール、10％2-メルカプトエタノール0.004％ブロモフェノールブルー、0.125 MトリスHCl pHは6.8）5分95ºCで、沸騰中の各核内画分の15μLを混ぜます。 20ミリアンペア（ミリアンペア）で、1.5時間ゲルと別々のタンパク質を変性10％ドデシル硫酸ナトリウム - ポリアクリルアミドゲル電気泳動（SDS-PAGE）にサンプルをロードします。 </li><li>ポリフッ化ビニリデン（PVDF）膜上に400ミリアンペアで1.5時間、エレクトロによってタンパク質を転送します。 </li><li> PBS中3％脱脂粉乳を使って室温で2時間ブロック膜。 </li><li> PBS / 0.3％の脱脂粉乳で1：500希釈でウイルスのE2A DNA結合タンパク質（B6-8 9）に対する一次抗体と4ºCでO / Nインキュベートします。 </li><li> 10分間、PBS / 0.1％Tween 20で洗浄膜3回。 </li><li>マウス抗IgG二ANを有する膜をインキュベートRTで2時間、PBS中の1：10,000希釈tibodyは1でホースラディッシュペルオキシダーゼ（HRP）に結合されました。 </li><li> PBSで膜を3回洗浄/ 0.1％のTween 20を10分間。 </li><li>増強化学発光及びX線フィルムを用いて膜を開発します。 </li></ol> RCF 4.ウイルスDNAの検出注：両方のNPLとRCF画分からのDNA単離のために、ステップ2.11で得られた全体積のRCFのために不良債権のために210μlの100μLを使用しています。 <ol><li>プロテイナーゼKを1mg / mlおよび0.5％Tween 20で55ºCで1時間サブ核画分をインキュベートします。 </li><li> 95℃で10分間、サンプルをインキュベートすることにより、プロテイナーゼKを不活性化します。 </li><li> 2分間室温で20,000×gで試料を遠心。 </li><li> SNを収集します。 3M酢酸ナトリウム1/10容量と1容量のイソプロパノールでDNAを沈殿させ、O / Nで4℃にて。 </li><li>室温で10分間20,000×gで試料を遠心。 </li><li> SN Uを破棄マイクロピペットを歌います。 20,000×gで70％エタノールと遠心で5分間、4°Cをペレットを洗浄します。 </li><li> 10mMトリス-塩酸pH7.4中の10μLに DNAを再懸濁</li><li> 260nmでの光学密度（OD）を測定することによって分光光度計を用いてDNAを定量化します。 </li><li>ウイルスDNAを増幅するためのヌクレオチド7273から、主要後期転写ユニット内の領域の増幅を可能にするプライマーを用いて反応容積の25μLでのTaq DNAポリメラーゼを使用する標準的PCR反応において、各核内画分からDNA 100ngのに使用するためにヌクレオチド7353（81塩基対）（FW：GAGCGAGGTGTGGGTGAGC; Rvの：GGATGCGACGACACTGACTTCA）。 95ºCで3分間の初期変性、20増幅（95ºC、72ºCで30秒間62ºCで1分間アニーリングおよび伸長で1分間）のサイクルとの最終的な伸長工程に続く次のサイクル条件を使用してください72ºCで3分間。 </li><li>エチジウム90 V.ステインで、2％アガロースゲルでPCR産物を実行します0.5μG / mlの最終濃度で臭化RCFでウイルスDNAの濃縮を裏付けるためにDNAを可視化します。細心の注意を払ってエチジウムブロマイドを処理し、常にこの化合物は強力な変異原であるように、手袋を着用してください。制度ガイドラインに従ってエチジウムブロマイドを廃棄してください。 </li></ol> RCF 5.後期ウイルスmRNAの検出注：両方のNPLとRCF画分からのRNAの単離のために、ステップ2.11で得られた総容量からRCFのために不良債権のために640μlの300μLを使用しています。 <ol><li>製造元の指示に従ったトリゾール使用して核内画分からRNAを分離します。 DEPC（diethilpyrocarbonate）処理水50μlの中のRNAを再懸濁します。 </li><li> （約3μgが得られた）、260 nmでODを測定するために分光光度計を用いてRNAを定量化します。 -70ºCで50 ngの/μlのアリコート中のRNAを保管してください。 </li><li>精製されたRNAをチェック5トータルRNAのngのステップ4.9に記載のサイクル条件を用いて、逆転写酵素（RT）の非存在下でのコントロールPCR反応を行うことによりDNA汚染の欠如のために。 <ol><li> DNAの混入がない場合何のアンプリコンが生成されるべきではありません。 DNA汚染が存在する場合は、37ºCで30分間、10 UのDNase I、RNase阻害剤の10 U、0.1 Mトリス-塩酸pHを8.3、0.5 M KCl及び15mMのMgCl 2を有する試料をインキュベートします。ステップ5.1のように、RNAを再単離するために進んでください。 </li></ol></li><li> RCFに関連するウイルスmRNAを分析するために、異なる遺伝子ファミリー（ 表1）から、アデノウイルス後期のmRNAを検出するために設計されたプライマーを用いたRT-PCRによって、各核内画分からRNA 100ngの増幅。 <ol><li>逆転写（RT）については、42ºCで1時間、70ºCで10分間反応容量の20μLの M-MuLVにRT酵素を用いた標準的なRT反応の各核内画分から、RNA 100ngのを使用しています。 </li><li>増幅のために、1を使用しますステップ4.9のように、PCR反応でcDNAのLをμ。 25サイクルの増幅に続いて95ºCで3分間の初期変性、（95ºCで1分間、アニーリング72ºCで30秒間、表1と拡張で指定された温度で1分間）し、次のサイクル条件を使用してください72ºCで3分間の最終伸長工程。 </li></ol></li><li> 0.5μグラム/ mlの最終濃度で、臭化エチジウム90 V.染色ゲルで2％のアガロースゲル中でRT-PCR産物を、実行し、RCFへのウイルス後期mRNAの関連を裏付けるために可視化します。ステップ4.10に示すようにエチジウムブロマイドを処理します。 </li></ol> RCFの6免疫蛍光可視化注：キャリーアウトは、この手順をクリーンベンチの下にほこり粒子とサンプルの汚染を避けるため、使用する前に、すべてのソリューションをフィルタリングします。 <ol><li>悲惨なステップ2.10で得られたRCF画分のスポット5μlのCTLYシランコートスライド上。 </li><li>室温で約5分間のスポット乾燥をしてみましょう。 </li><li>再水和物をゆっくりと間接的にRCFスポットの横にあるドロップでのPBS 500μLをピペッティングして、スライドを傾けることによって流れせることによって。側から過剰のPBSを切ります。 </li><li> RTで2時間、PBS / 5％のウシ血清アルブミン（BSA）の500μLに試料を覆うことによりブロック。 </li><li>穏やかに間接的に試料上にPBSを500μリットルをピペットでスライドを3回洗浄します。過剰のPBSを切るためにスライドを傾けます。 </li><li>室温で2時間、PBS中で1：500希釈でウイルスE2A DNA結合タンパク質（B6-8 9）に対する一次抗体とサンプルをインキュベートします。抗体溶液の20μLにスポットされたサンプルをカバーし、乾燥を避けるために、湿ったチャンバー内でインキュベートします。 </li><li>穏やかに間接的に試料上にPBSを500μリットルをピペッティングすることにより、PBS / 0.02％Tween 20でサンプルを3回洗浄します。元を切るためにスライドを傾けてセス洗浄溶液。 </li><li> 4ºCで1時間、PBS中で希釈2,000：1で、488 nmで励起される蛍光色素分子に結合されたマウス抗IgG二次抗体を用いて、サンプルをインキュベートします。抗体溶液の20μLにスポットされたサンプルをカバーし、乾燥を避けるために、湿ったチャンバー内でインキュベートします。 </li><li>穏やかに間接的に試料上にPBSを500μリットルをピペッティングすることにより、PBS / 0.02％Tween 20でサンプルを3回洗浄します。過剰の洗浄溶液を切るためにスライドを傾けます。 </li><li>メディアをマウントするように2μリットルのPBS / 10％グリセロールを使用したカバーガラスとスライド上にスポットしたサンプルをカバーしています。明確なマニキュアで密封し、アルを保存-20ºC。 </li><li>波長488nmでの63X対物1.4開口数（NA）と、蛍光顕微鏡を用いてサンプルを分析します。 </li></ol>

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The authors have nothing to disclose.

In order to elucidate the molecular mechanisms that govern regulation of cellular activities by viral infection understanding the composition and activities associated with RC would be instrumental. Therefore, to make a detailed analysis of RC, we established a cell-free system that takes advantage of the size and biochemical composition of these virus-induced structures, to isolate subnuclear fractions enriched with RC using a simple procedure that relies on velocity gradients with sucrose cushions. Critical steps of th...

アデノウイルスは、感染細胞の核内で複製する二本鎖DNAゲノムを含みます。ウイルスDNAは核に入ると、それは、PML核小体1に隣接して局在します。ウイルスの初期遺伝子発現に続いて、核アーキテクチャは劇的と呼ばれる、ウイルスの微小環境の形成を誘導し、再編成されたウイルス複製のコンパートメント（RC）2。アデノウイルス（AD）RCは、ウイルスゲノムの複製およびウイルス後期遺伝子の発現が起こる部位であるので、これらのプロセスに関与するすべての必要なウイルスおよび細胞因子の動員のための環境を提供します。興味深いことに、このようなDNA損傷応答等の細胞抗ウイルス応答を担う細胞タンパク質、種々の、先天性免疫応答および腫瘍抑制は、これらのウイルスの部位2に同時オプトインしています。同時に調整しながらしたがって、広告RCは、効率的なウイルス複製を促進する調節ハブを考慮することができますこれらの構造は、ウイルス - 宿主細胞の相互作用を理解するための鍵であることを示す細胞の抗ウイルス応答、。それにもかかわらず、RC形成の分子機構は、それらの組成および関連する活動があまり理解されていません。 核内で複製する他のDNAウイルスからのアデノウイルスのRCと同様に、RCが細胞質RC 3とは対照的に、膜に関連付けられていません。また、これらのウイルス誘発性構造は、タンパク質および核酸の完全に構成される可能性があります。 RNAウイルス（通常ウイルス工場と呼ばれる）に感染した細胞内に形成されたRCは、その詳細な形態的、機能的および生化学的特徴4が容易になったそれらの細胞質局在性および膜結合状態、を利用して、単離されています。 我々の知る限り、核ウイルスRCはおそらく核アーキテクチャの複雑さと核内メートルがないため、単離されていませんそれらの単離を容易にするであろうembranes。彼らの研究は、免疫蛍光顕微鏡、魚や透過型電子顕微鏡で代わりに依存してきました。しかし、核内構造体を単離することに固有の合併症にもかかわらず、このような核小体とカハール体として他の核ドメインが5,6の前に単離されています。核小体とRCの両方がタンパク質や核酸で構成され、0.5の間の直径持っているので - 5μmで、私たちは、RCはまた、分離に適してなければならないという仮説を立てました。そのため、より正確にはRCに関連する分子組成および機能を特徴付けるために、我々は、RCで強化された核内画分を単離するための新規な方法を確立しました。この目的のために、我々は核 ​​小体7または他の核ドメイン6を単離するために使用される手順と同様の速度勾配およびスクロースクッションを使用して、サブ核画分を調製して、分子組成の研究との関連活動を可能にする無細胞系を確立しましたRC。この技術は、したがって、ウイルス - 宿主細胞相互作用の理解を進め、また、核内で複製する他のウイルスからのRCの詳細な分析を容易にし、アデノウイルス内に形成されたものと同様の寸法の複製区画の形成を誘導するはずである強力なツールを表しているべきですこのような、ヘルペスウイルス、パピローマウイルスまたはポリオーマウイルスなどの感染細胞、。

<table border="0">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>12100-046</td>
			<td>Warm in 37 &#186;C water bath before use</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>12484-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose, Ultra Pure</td>
			<td>Research Organics</td>
			<td>0928S</td>
			<td>Prepare a 2.55 M stock solution and store at 4 &#186;C</td>
		</tr>
		<tr>
			<td>Dounce homogenizer</td>
			<td>Kontess Glass Company</td>
			<td>884900-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Branson 1800 Ultrasonic Bath</td>
			<td>Branson</td>
			<td>Z769533&#160;SIGMA</td>
			<td>Turn on 15 min before use.</td>
		</tr>
		<tr>
			<td>Peroxidase AffiniPure F(ab&#39;)&#8322; Fragment Goat Anti-Mouse IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-036-003</td>
			<td>Use at a 1:10,000 dilution in PBS/0.03% non-fat milk</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG1 Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td>Life Technologies</td>
			<td>A-21121</td>
			<td>Use at a 1:2,000 dilution in PBS</td>
		</tr>
		<tr>
			<td>Silane-Prep Slides</td>
			<td>Sigma</td>
			<td>S4651-72EA</td>
			<td>Open in a laminar flow cabinet</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico Chemiluminescent Substrate</td>
			<td>Pierce ThermoScientific</td>
			<td>34080</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of viral replication compartment-enriched sub-nuclear fractions from adenovirus-infected normal human cells

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.

ウイルス複製区画（RC）は、他の核内ドメインに類似したタンパク質および核酸からなる核内のウイルスによって誘発される構造であるので、それらは、生化学的特徴に基づいて速度勾配によって分離を受けやすいことが判明しました。分画プロトコルにおける重要なステップは、サンプルは、異なる細胞下分画の完全性を保証するために、明視野顕微鏡法によって監視される必要がある各?...

Watch this Scientific Journal Video about Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells at JoVE.com

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.

アデノウイルスに感染したヒト正常細胞からのウイルス複製コンパートメントに富むサブ核画分の単離

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

isolation-viral-replication-compartment-enriched-sub-nuclear

Research

JoVE Journal

Immunology and Infection

8.7K ビュー.  Universidad Autónoma del Estado de Morelos. We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC. 

ビデオ: Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

ウイルスの変異頻度のRNAウイルスと特性の忠実性の変異の分離

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

免疫・感染学

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

ヌクレオカプシドからウイルスDNAの調製

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

呼吸器病原体に対する宿主細胞応答を研究するために偏ヒト気道上皮Calu-3細胞の液体に覆われた文化の確立<em&gt;インビトロ</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

ヒト胸腺からの骨髄樹状細胞と上皮細胞の単離

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Jsubtomoを用いた電子Cryotomography再建からのウイルスのエンベロープ糖タンパク質スパイクの平均化

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

ヒト母体·胎児のインターフェイスから白血球の単離

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

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 ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

表面上のヒトノロウイルスの検出のためのスワブサンプリング方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

人間の女性の生殖器官から樹状細胞の表現型および機能研究の分離

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

In Vitro Differentiation Model of Human Normal Memory B Cells to Long-lived Plasma Cells

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or mucosa and ensure humoral immunity. Due to their low frequency and location, the study of PCs is difficult in human. We reported a B to PC in vitro differentiation model using selected combinations of cytokines and activation molecules that allow to reproduce the sequential cell differentiation occurring in vivo. In this in vitro model, memory B cells (MBCs) will differentiate into pre-plasmablasts (prePBs), plasmablasts (PBs), early PCs and finally, into long-lived PCs, with a phenotype close to their counterparts in healthy individuals.&#160;We also built an open access bioinformatics tools to analyze the most prominent information from GEP data related to PC differentiation. These resources can be used to study human B to PC differentiation and in the current study, we investigated the gene expression regulation of epigenetic factors during human B to PC differentiation.

Using multi-step culture systems, we report an in vitro B cell to plasma cell differentiation model.

通常記憶 B 細胞の長命の形質細胞への分化モデル

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or ...