私たちは、原稿の準備プロセスを通して彼の洞察とアドバイスをケンNealsonに感謝したいと思います。

1。ウイルス懸濁液の調製フローサイトメトリーを経由して、単一のビリオンの分離前に、固定されていないウイルスの懸濁液を調製。 <ol><li>必ず最後のウイルス懸濁液を作る以前に確立されたプロトコルを使用してウイルス粒子を分離する0.1μmのフィルターTE（トリス-EDTA、pHが8）に含まれています。我々は、他の方法が使用化学凝集7が開発されているが（TFF）6に近づくタンジェンシャルフローろ過の使用をお勧めします。 </li><li>落射蛍光8-10またはフローサイトメトリー11,12を使用して確立されたプロトコルを用いて、 例えば 、ウイルス粒子を列挙する。 </li><li> 2エッペンドルフチュー​​ブ（最終容量は1,000μLであることが推奨されます）にアリコートウイルス懸濁液。 </li></ol> 2。フローサイトメトリー<ol><li>カスタム前方散乱PMT（FSC PMT）を搭載したBD FACSAria IIフローサイトメーターを使用してウイルスをソートするには、0.1μmのフィルターTE、pHを8にウイルス粒子を希釈する。200イベント秒-1のイベント発生率のための適切な力価を0。 </li><li>信号対雑音配給などを最大化するためにしきい値を設定する。 T4とラムダファージ粒子、次を含む混合群集のために推奨されています：FSC PMTを1,000とSSCで200。 </li><li>唯一の1を含んでいる &quot;空白&quot;サンプル）0.1μmのフィルターTEおよび2）1E-4で0.1μmのフィルターTEとSybrGreen1株式の希釈（10,000 X）を100μlを実行します。 </li><li>汚されたウイルス懸濁液（株式の1E-4希釈でSybrGreen1）、5,000イベントそれぞれの合計にウイルス粒子が続くバックグラウンドを評価するための染色ウイルス懸濁液の小アリコート（100μlをお勧めします）を実行します。これは、ソート前に、必要に応じて、濃度を確認し、選別ゲートを調整することである。私たちは、ウイルス粒子の中心にタイトなゲートをお勧めします。また、ゲートを生成するために私たちは、SYBRグリーンとFSC PMTプロットを使用しています。 </li><li> 1％低融点（LMP）の5μlをアガロース（TBEバッファ内の）それぞれに37℃に冷却し、よくポリテトラフルオロエチレン（PTFE）顕微鏡スライド上（電子顕微鏡科学）。 </li><li>並べ替えウイルス粒子を捕捉するためにフローサイトメーターにPTFEの顕微鏡スライドをロードします。 </li><li>直接LMPアガロースとPTFEスライド上にSYBR緑ステンドウイルス粒子のようなものを開始します。我々は、共焦点レーザー走査顕微鏡（CSLM）中のウイルスの深さの検出を助けるために、いくつかのウェル内の10以上のイベント（ウイルス粒子）の並べ替えをお勧めします。 </li><li>ソートが完了すると、フローサイトメーターからスライドを削除し、追加5μlをよりLMPアガロースしたがって、ウイルス粒子（s）を埋め込み、各ウェルに37℃に冷却した。 </li></ol> 3。共焦点顕微鏡を使用して単一のビリオンの可視化単一ビリオンが所望される場合、各ウェルの個々のウイルスが含まれているかどうかを決定することは不可欠であるとCLSMは、単一の粒子が存在し、その複数のウイルスであることを確認するために3Dに埋め込まれたビリオン（s）を可視化することが必要である互いの上に積層されていない。 <ol><li>波長488 nmの励起顕微鏡レーザーを設定します。 </li><li> 63X長作動距離対物レンズを用いて埋め込まれたウイルス粒子のイメージ。 </li></ol> 4。全ゲノム増幅<ol><li>単一のウイルスによるウェルをCLSMで識別された後、アガロース（ 上皮内 ）内で複数の置換増幅（MDA）を使用して、全ゲノム増幅を行う。 </li><li> DNA汚染を導入する可能性を減少させるために、スライドから所望のアガロース &#39;ビーズ&#39;を削除し、滅菌PCRチューブに入れた。滅菌ピンセットおよび/またはこのステップのカミソリの刃のペアを使用してください。 </li><li>サーモサイクラーのヒートブロックで3分間熱（94℃）を使用して、その場でウイルス粒子を溶解。 </li><li>以下の変更を加えて、製造業者の推奨に従ってMDA反応（GenomiPhi HYキット、GEヘルスケア）の実行<ol><li>サンプルと反応BUFの量を減らす30 11.25μLとインキュベートするFERS°非特異的増幅を低減するために2時間の最大のためのC。 </li></ol></li><li> 1.7ミリリットルエッペンドルフチュー​​ブにゲノムDNA（gDNAを）を転送することにより、増幅されたゲノム物質を浄化し、TBE緩衝液中で1/10量の3M酢酸ナトリウム（NaOAcを）（LMPアガロースの調製に使用したのと同じ緩衝液）を追加する。 </li><li> 55時場所サンプル（秒）°アガロースを溶解するために10分間。 </li><li>管（群）42に移動°C、酵素を加える前に温度を低下させる。 </li><li>各チューブへのβ-アガラーゼ2台を追加し、2時間インキュベートする。 </li><li>チューブにバッファー飽和フェノール溶液1ボリュームを追加し、精力的に混合し、15分間3,500 xgで遠心。新しい1.7ミリリットルエッペンドルフチュー​​ブに上清を含むDNAを転送します。 </li><li> Glycoblueの100％イソプロパノール及び1μlののつのボリュームを追加します。反転チューブでよく混ぜる。 </li><li> 60分間4℃で28,000×gで遠心分離します。デカントイソプロパノールペレットを乱さないように確認すること。 70％ETHA150μlのを追加各チューブにNOL。 10分間28,000 xgで、室温でスピン。エタノールをデカントし、空気はDNAペレットを乾燥し、TEバッファーの適切な量でDNAを再懸濁します。 </li><li>我々は、次の変更を加えて手順4.4から4.9を繰り返してお勧めします。 3-5複製MDA反応を設定し、1時間に30℃でのインキュベーションを減らす。所望の濃度を得るために、95〜100％のエタノールを用いて沈殿物を精製し、続いてサンプルをプール。 </li></ol> 5。代表的な結果 図1は、SVGのプロセスをまとめたものです。これらの方法は、配列決定のために十分であるのgDNA量を得るために、MDA、続いて混合された集合体から単一のビリオンを分離するためにアガロースを含有するPTFE顕微鏡スライド上にウイルス粒子をソートするフローサイトメトリーを使用する。 概念実証として、バクテリオファージラムダおよびT4を混合し、SVGプロセス11に供した。ビリオンをアガロース、CLSMワシントン州でキャプチャされ、埋め込まれていた後はsは視覚化し、単一ビリオンが得られたことを確認するために使用される、 単一のビリオンとウェルが同定されたら、その結果を図2に示されているが、それらは、MDAのgDNAのために選択した。私たちは、その後、T4と孤立ビリオン、 図3。識別するラムダ孤立ファージラムダがゲノム解読のために選択されたためのマルチプレックスPCRは、特定の使用。 ゲノム配列決定は、SVGを用いて得られた被覆のレベルを識別するためにマッピングを参照するために行った454-チタン技術および配列読み取りを行った。 図4のほぼ全体ラムダゲノム（第5塩基対を除いて）を回収したこと。 <img alt="図1" src="/files/ftp_upload/3899/3899fig1.jpg" /> 図1。混合群集を含むSVG方法論。ウイルス懸濁液は、その後のあるフローサイトメトリーを経由して、単一のウイルス粒子に還元される個人アガロース &quot;ビーズ&quot;にorted。ウイルスはアガロースポストフローサイトメトリーソーティングの追加レイヤを重ねることによってアガロースビーズ内に埋め込まれます。最後に、全ゲノム増幅をin situで行われる。 <img alt="図2" src="/files/ftp_upload/3899/3899fig2.jpg" /> 図2。共焦点レーザーは、アガロースビーズに埋め込 ​​ま並べウイルス粒子の顕微鏡写真をスキャン。）アガロースビーズ内のSYBR Green I染色ウイルス粒子の3次元再構築を。挿入図：絶縁ウイルス粒子の高倍率アガロースビーズ内ステンドシングルウイルス粒子の相対的な蛍光のB）プロファイルプロット。 <img alt="図3" src="/files/ftp_upload/3899/3899fig3.jpg" /> 図3。 PCRを用いたバクテリオファージの識別。）&lt;1：/ strong&gt;のマルチプレックスPCRは、遺伝子型にレーンをT4とラムダ特異的プライマーを用いた。 TrackIt 1キロバイトプラスラダー（Invitrogen社）、2。ラムダインテグラーゼ（750塩基対）、3。 T4主要キャプシドタンパク質（1,050 BP）、4。 1：ラムダインテグラーゼおよびT4主要キャプシドタンパク質のミックスB）追加の座とその後のラムダ特異的PCRはさらにファージゲノムの分離、レーンを確認する。ラムダインテグラーゼ（750塩基対）、2。ラムダリプレ​​ッサー（356塩基対）3。ラムダSIE（重複感染の排除）（456塩基対）4。 TrackIt 1キロバイトプラスラダー（Invitrogen社）。 <img alt="図4" src="/files/ftp_upload/3899/3899fig4.jpg" /> 図4。ラムダゲノム属性と報道。）GCプロット、ラムダのB）ゲノムマップ（から適応<a href="http://img.jgi.doe.gov" target="_blank">http://img.jgi.doe.gov</a> ）、およびC）SVGのマッピング参照バクテリオファージλ、x軸に読み込んでゲノム位置、屋軸ロボットPは％カバレッジです。

<ol>
	<li>Raghunathan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+DNA+amplification+from+a+single+bacterium.">Genomic DNA amplification from a single bacterium.</a> Appl. Environ. Microbiol. 71, 3342-337 (2005).</li><li>Lasken, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+genomic+sequencing+using+Multiple+Displacement+Amplification.">Single-cell genomic sequencing using Multiple Displacement Amplification.</a> Curr. Opin. Microbiol. 10, 510-516 (2007).</li><li>Ishoey, T., Woyke, T., Stepanauskas, R., Novotny, M., Lasken, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+sequencing+of+single+microbial+cells+from+environmental+samples.">Genomic sequencing of single microbial cells from environmental samples.</a> Curr. Opin. Microbiol. 11, 198-204 (2008).</li><li>Edwards, R. A., Rohwer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+metagenomics.">Viral metagenomics.</a> Nature Reviews Microbiology. 3, 504-510 (2005).</li><li>Rohwer, F., Prangishvili, D., Lindell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+viruses+in+the+environment.">Roles of viruses in the environment.</a> Environ. Microbiol. 11, 2771-2774 (2009).</li><li>Williamson, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Sorcerer+II+Global+Ocean+Sampling+Expedition:+metagenomic+characterization+of+viruses+within+aquatic+microbial+samples.">The Sorcerer II Global Ocean Sampling Expedition: metagenomic characterization of viruses within aquatic microbial samples.</a> PLoS ONE. 3, e1456 (2008).</li><li>John, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+efficient+method+for+concentration+of+ocean+viruses+by+chemical+flocculation.">A simple and efficient method for concentration of ocean viruses by chemical flocculation.</a> Environ. Microbiol. Rep. 3, 195-202 (2011).</li><li>Noble, R., Fuhrman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+SYBR+Green+I+for+rapid+epifluorescence+counts+of+marine+viruses+and+bacteria.">Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria.</a> Aquat Microb Ecol. 14, 113-118 (1998).</li><li>Hara, S., Terauchi, K., Koike, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abundance+of+viruses+in+marine+waters:+assessment+by+epifluorescence+and+transmission+electron+microscopy.">Abundance of viruses in marine waters: assessment by epifluorescence and transmission electron microscopy.</a> Appl. Environ. Microbiol. 57, 2731-2734 (1991).</li><li>Hennes, K. P., Suttle, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+counts+of+viruses+in+natural+waters+and+laboratory+cultures+by+epifluorescence+microscopy.">Direct counts of viruses in natural waters and laboratory cultures by epifluorescence microscopy.</a> Limnol. Oceanogr. 40, 1050-1055 (1995).</li><li>Marie, D., Brussaard, C. P. D., Thyrhaug, R., Bratbak, G., Vaulot, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+marine+viruses+in+culture+and+natural+samples+by+flow+cytometry.">Enumeration of marine viruses in culture and natural samples by flow cytometry.</a> Appl. Environ. Microbiol. 65, (1999).</li><li>Brussaard, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+bacteriophages+using+flow+cytometry.">Enumeration of bacteriophages using flow cytometry.</a> Methods Mol. Biol. 501, 97-111 (2009).</li><li>Allen, L. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Virus+Genomics:+A+New+Tool+for+Virus+Discovery.">Single Virus Genomics: A New Tool for Virus Discovery.</a> Plos One. 6, (2011).</li><li>Hutchison, C. A., Smith, H. O., Pfannkoch, C., Venter, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+cloning+using+phi+29+DNA+polymerase.">Cell-free cloning using phi 29 DNA polymerase.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 17332-17336 (2005).</li></ol>

利害の衝突は宣言されていない。

SVGの手法を適用する際に重要な多くの要因を考慮しなければならない。ジェノタイピングは、概念実証実験13の間に行われたように、保存されたプライマーはすべてのウイルスのグループ間で利用できないなどの環境や未知の分離のための有効なオプションではありません。また、バックグラウンドDNA合成又は非特異的な増幅は、一般的にMDA反応14を用いて増幅中に報告され?...

<table border="1">
	<tbody>
		<tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>1X TE buffer</td>
			<td>Invitrogen</td>
			<td>12090-015</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Buffer-saturated Phenol</td>
			<td>Invitrogen</td>
			<td>15513-039</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>GenomiPhi HY kit</td>
			<td>GE Healthcare</td>
			<td>25-6600-22</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glycoblue</td>
			<td>Invitrogen</td>
			<td>AM9516</td>
			<td>15 mg/ml</td>
		</tr>
		<tr>
			<td>LMP agarose</td>
			<td>Invitrogen</td>
			<td>16520100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PTFE microscope slide</td>
			<td>Electron Microscopy Sciences</td>
			<td>63430-04</td>
			<td>24 well, 4 mm Diameter</td>
		</tr>
		<tr>
			<td>SybrGreen</td>
			<td>Invitrogen</td>
			<td>S7585</td>
			<td>10,000X</td>
		</tr>
		<tr>
			<td>&beta;-agarase</td>
			<td>New England Biolabs</td>
			<td>M0392S</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

isolation and genome analysis of single virions using 'single virus genomics'

単一の微生物細胞の全ゲノム増幅および配列決定は、栽培1-3を必要とせずにゲノム特性評価を可能にします。ユビキタスと私たちの惑星4上で最も数多くのエンティティとすべての環境5で重要であるウイルスは、同様のアプローチを介して明らかにされていない。ここでは、 &#39;シングルウイルスゲノミクス&#39;（SVG）と呼ばれる単一のビリオンのゲノムを単離し、特徴付けるためのアプローチについて説明します。 SVGは、その後の配列決定反応に用いることができる高分子量のゲノムDNA（gDNAを）を得るために、個々のウイルスおよび全ゲノム増幅を分離するためにフローサイトメトリーを利用する。

Watch this Scientific Journal Video about Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics' at JoVE.com

Isolation and Genome Analysis of Single Virions using &#039;Single Virus Genomics&#039;

単一のウイルスゲノム（SVG）は、単一のビリオンのゲノムを分離して増幅する方法である。混合された集合体のウイルス懸濁することにより、ビリオンを捕捉し、下流の処理中にゲノムせん断を低減、アガロースを含む個別のウェルを有する顕微鏡スライド上にフローサイトメトリーを用いてソートされる。全ゲノム増幅は、配列決定のために適しているゲノム材料をもたらす複数の置換増幅（MDA）を用いて達成される。

&#39;シングルウイルスゲノミクス&#39;を使用してシングルビリオンの単離とゲノム解析

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

isolation-genome-analysis-single-virions-using-single-virus

Research

JoVE Journal

Immunology and Infection

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

10.8K ビュー.  The J. Craig Venter Institute. 単一のウイルスゲノム（SVG）は、単一のビリオンのゲノムを分離して増幅する方法である。混合された集合体のウイルス懸濁することにより、ビリオンを捕捉し、下流の処理中にゲノムせん断を低減、アガロースを含む個別のウェルを有する顕微鏡スライド上にフローサイトメトリーを用いてソートされる。全ゲノム増幅は、配列決定のために適してい?...

ビデオ: Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

に関与する宿主因子のためのRNAiスクリーニングワク</em使用して>ウイルス感染ショウジョウバエセル

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

免疫・感染学

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

ハイドロキシアパタイトクロマトグラフィーを用いた環境ウイルスのコミュニティから一本鎖DNA、二本鎖DNAとRNAの分離

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

の単一細胞解析枯草菌</em蛍光顕微鏡とフローサイトメトリーを用いて>バイオフィルム

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures.

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

光活性化単一分子追跡を使用してライブ細菌細胞におけるタンパク質-DNA相互作用を可視化する

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome ...

Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and epidermis home a variety of immune cells in both homeostatic and inflammatory conditions. Tools to obtain skin cell release for cytofluorimetric analyses are, therefore, very useful in order to study the complex network of immune cells residing in the skin and their response to microbial stimuli. Here, we describe an efficient methodology for the digestion of mouse skin to rapidly and efficiently obtain single-cell suspensions. This protocol allows maintenance of maximum cell viability without compromising surface antigen expression. We also describe how to take and digest skin samples from different anatomical locations, such as the ear, trunk, tail, and footpad. The obtained suspensions are then stained and analyzed by flow cytometry to discriminate between different leukocyte populations.

The skin is home to a complex immune cell network. We describe an efficient methodology for the digestion of mouse skin, from different parts of the animal's body, in order to obtain a single-cell suspension and analyze the different leukocyte populations resident in the skin by flow cytometry.

別のマウス皮膚の領域から細胞蛍光分析のための単一細胞懸濁液の調製

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

治療薬にリンパ腫細胞の感度での役割を決定するために、好中球の単離と解析

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

madSTORMを用いたT細胞の高度に多重化された超解像イメージング

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

イムノフェノ タイプと大量のフローサイトメトリーによる末梢血中のサイトカイン産生の単一細胞解析

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

下流の単一細胞分析のため尿路感染症のマウスモデルから生成された単一細胞内細菌群集の分離

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This technique allows for probing interaction strengths and dynamics on a live cell membrane, including those between cells, receptor and ligands, and alongside many other variations. It also works as a mechanism to deliver a physical or biochemical stimulus on single cells in a spatiotemporally controlled manner, thus allowing specific cell activation and subsequent cellular events to be monitored in real-time when combined with live-cell fluorescence imaging. The key step in those AFM-SCFS measurements is AFM-cantilever functionalization, or in other words, attaching a subject of interest to the cantilever. Here, we present methods to modify AFM cantilevers with a single T cell and a single polystyrene bead respectively for immunological studies. The former involves a biocompatible glue that couples single T cells to the tip of a flat cantilever in a solution, while the latter relies on an epoxy glue for single bead adhesion in the air environment. Two immunological applications associated with each cantilever modification are provided as well. The methods described here can be easily adapted to different cell types and solid particles.

We present a protocol to functionalize atomic force microscope (AFM) cantilevers with a single T cell and bead particle for immunological studies. Procedures to probe single-pair T cell-dendritic cell binding by AFM and to monitor the real-time cellular response of macrophages to a single solid particle by AFM with fluorescence imaging are shown.

免疫学的単一細胞力分光法のための単一T細胞または単一粒子による原子間力顕微鏡片持ちの機能化

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This ...