우리는 원고 준비 과정을 통해 자신의 통찰력과 조언 켄 Nealson에게 감사의 말씀을 전합니다.

1. 바이러스 정학의 준비 유동 세포 계측법을 통해 단일 ​​비리의 분리하기 전에, 고정되지 않은 바이러스 현탁액을 준비합니다. <ol><li> 반드시 최종 바이러스 현탁액을 만들기 이전에 설정된 프로토콜을 사용하여 바이러스 입자를 분리하는 것은 0.1에 포함되어 μm의 여과 TE (트리스 - EDTA (에틸렌 다이아 민 테트라 초산), 산도 8). 다른 방법은 해당 사용 화학 응집 7 개발되어 있지만, 우리는 접근 접선 흐름 여과 (TFF)를 사용하여 6을 추천합니다. </li><li> 예를 들어, epifluorescence에 8-10 또는 유동 세포 계측법 11,12을 사용하여 설정 프로토콜을 사용하여 바이러스 입자를 열거합니다. </li><li> 두 에펜 도르프 튜브 (최종 부피는 1,000 μL 할 것을 권장합니다)로 나누어지는 바이러스 현탁액을. </li></ol> 2. 유동 세포 계측법 <ol><li> 사용자 정의 포워드 분산 PMT (FSC PMT)가 장착 BD FACSAria II 흐름 cytometer에를 사용하여 바이러스를 정렬하려면, 0.1에서 바이러스 입자를 희석 TE, pH가 8 μm의 여과.0 200 이벤트 초 -1의 경우 속도에 대한 적절한 역가합니다. </li><li> 신호 대 잡음 식량을 극대화하기 위해 임계 값, 예를 들어 설정합니다. T4 및 람다 파지 입자에게 다음을 포함하는 혼합 조립을 권장합니다 : FSC PMT를 1,000 SSC에서 200. </li><li> 단지 1을 포함하는 &quot;빈&quot;샘플) 0.1 μm의 필터 TE 2) 1E-4 0.1 μm의 필터 TE와 SybrGreen1 주식의 희석 (10,000 X)의 100 μl를 실행합니다. </li><li> 스테인드 바이러스 현탁액 (주식 1E-4 희석 SybrGreen1), 5000 사건 각의 총에 바이러스 입자 뒤에 배경을 평가하는 흠 바이러스 현탁액의 작은 나누어지는 (100 μl를 권장합니다)를 실행합니다. 이 종류에 앞서, 필요한 경우, 농도를 확인하고 정렬 게이트를 조정하는 것입니다. 우리는 바이러스 입자의 중심에 꽉 게이트를 추천합니다. 또한, 문을 생성하기 위해 우리는 SYBR 녹색과 FSC PMT 플롯을 사용합니다. </li><li> 1 % 낮은 녹는 점 (LMP)의 5 μl를 추가 아가로 오스 (TBE 버퍼)에 각각 37 ° C로 냉각또한 폴리 테트라 플루오로 에틸렌 (PTFE) 현미경 슬라이드 (전자 현미경 과학). </li><li> 정렬 된 바이러스 입자를 캡처하는 흐름 cytometer에에 PTFE 현미경 슬라이드를로드합니다. </li><li> 직접 LMP 아가로 오스와 PTFE 슬라이드에 SYBR 녹색 스테인드 바이러스 입자의 정렬을 시작합니다. 우리는 공 촛점 레이저 스캐닝 현미경 (CSLM) 동안 바이러스의 깊이를 감지에 도움이 몇 가지 우물에서 10 개 이상의 이벤트 (바이러스 입자를) 정렬 좋습니다. </li><li> 정렬이 완료되면, 흐름 cytometer에에서 슬라이드를 제거하고 추가 5 μL 더 LMP 아가 따라서 바이러스 성 입자 (들)을 포함, 각 well에 37 ° C로 냉각. </li></ol> 3. 공 촛점 현미경을 사용하여 단일 비리를 시각화 하나의 virion의가 필요한 경우 각도 각 바이러스를 포함하는 경우, 그 결정은 필수적이며, CLSM은 하나의 입자가 존재하고 있다는 것을 확인하는 차원에 포함 된 virion의 (들)을 시각화하는 데 필요한 여러 바이러스서로 위에 쌓인되지 않습니다. <ol><li> 파장 488 nm의 여기에 현미경 레이저를 설정합니다. </li><li> 63X 긴 작동 거리 목표를 사용하여 포함 된 바이러스 입자 이미지. </li></ol> 4. 전체 게놈 증폭 <ol><li> 하나의 바이러스에 우물이 CLSM으로 식별되면, 아가로 오스 (현장에서) 내에서 여러 변위 증폭 (MDA)를 사용하여 전체 게놈 증폭을 수행합니다. </li><li> DNA 오염을 도입의 가능성을 줄이려면, 슬라이드에서 원하는 아가 &#39;구슬&#39;을 제거하고 멸균 PCR 튜브에 배치. 멸균 핀셋 및 / 또는이 단계 면도날의 쌍을 사용합니다. </li><li> 현장이 thermocycler의 열 블록에서 3 분 (94 ° C) 열 사용에 바이러스 입자를 용해. </li><li> 다음과 같이 수정하여 MDA 반응 다음과 같은 제조업체의 권장 사항 (GenomiPhi HY 키트, GE 헬스 케어)을 수행 <ol><li> 시료와 반응 BUF의 볼륨을 줄입니다30 11.25 μL와 부화에 fers ° 비특이적 증폭을 줄이기 위해 2 시간의 최대 C. </li></ol></li><li> 1.7 ML 에펜 도르프 튜브에 게놈 DNA (gDNA를)를 전송하여 증폭 게놈 자료를 순화하고 1 / 10 양 TBE 버퍼에 3 M의 아세트산 나트륨 (NaOAc () 같은 버퍼 LMP 아가로 오스 준비에 사용)를 추가합니다. </li><li> 55 장소 샘플 (가) ° C 10 분 동안 아가로 오스를 분해한다. </li><li> 42 튜브 (들)을 이동 ° C 효소를 추가하기 전에 온도를 줄일 수 있습니다. </li><li> 각각의 튜브와 2 시간 동안 배양 β-agarase 2 개를 추가합니다. </li><li> 튜브 버퍼 포화 페놀 용액 1 볼륨을 추가하고, 적극적으로 혼합하고 15 분 3,500 XG에 원심 분리기. 새로운 1.7 ML 에펜 도르프 튜브에 뜨는을 포함하는 DNA를 전송합니다. </li><li> Glycoblue 100 % 이소프로판올 및 1μL의 하나의 볼륨을 추가합니다. 반전 튜브로 잘 섞는다. </li><li> 60 분 동안 4 ° C에서 28,000 XG에 원심 분리기. 가만히 따르다 이소프로판올 펠렛을 방해하지 않도록하고. 70 % 에탄올 150 μl를 추가각 튜브 NOL. 10 분 동안 28,000 XG와 RT에서 회전. 에탄올을 가만히 따르다, 공기는​​ DNA 펠렛을 건조 TE 버퍼의 적절한 볼륨 DNA를 resuspend을. </li><li> 우리는 다음과 같은 수정을 단계 4.4-4.9를 반복 좋습니다. 3-5 복제 MDA 반응을 설정하고 1 시간에 30 부화 ° C를 줄일 수 있습니다. 원하는 농도를 얻기 위해 95~100% 에탄올을 사용하여 정화 및 침전 후 풀 샘플입니다. </li></ol> 5. 대표 결과 그림 1은 SVG 과정을 요약 한 것입니다. 이러한 방법은 순서에 충분 gDNA를의 수량을 얻기 위해 MDA 다음에 혼합 조립에서 하나의 바이러스 입자를 분리하는 아가로 오스를 포함하는 PTFE 현미경 슬라이드에 바이러스 입자를 정렬하는 유동 세포 계측법을 사용합니다. 증거의 개념으로, 박테리오파지 람다 및 T4는 혼합 및 SVG 과정 11 대상. 비리는 아가, CLSM 워싱턴에서 캡처 및 임베디드되자의 시각화 및 단일 virion의를 얻을되었는지 확인하는 데 사용;. 결과는 그림 2에 표시되면 하나의 비리와 우물이 확인되었다, 그들은 gDNA를의 MDA으로 선택 하였다. 우리는 T4와 격리 virion의 그림 3. 식별 할 람다 고립 된 파지 람다는 게놈 시퀀싱 선정을위한 멀티 플렉스 PCR은 특정 사용됩니다. 게놈 시퀀싱은 SVG를 사용하여 얻은 범위의 수준을 식별하는 매핑을 참조 할 대상이 된 454 티타늄 기술과 연속 읽기를 사용하여 수행 하였다. 그림 4는 거의 모든 람다 게놈 (제 5 BP의 예외) 복구되었다. <img alt="그림 1" src="/files/ftp_upload/3899/3899fig1.jpg" /> 그림 1. 혼합 조립을 포함하는 SVG 방법론. 바이러스 현탁액은 다음의 아르 유동 세포 계측법을 통해 하나의 바이러스 입자로 감소개별 아가 &quot;비즈&quot;에 orted. 바이러스는 아가로 오스 게시물 유세포 정렬의 추가 레이어와 중첩하여 아가 로스 비드 내에 포함되어 있습니다. 마지막으로, 전체 게놈 증폭 현장에서 수행됩니다. <img alt="그림 2" src="/files/ftp_upload/3899/3899fig2.jpg" /> 그림 2. 공 초점 레이저 아가로 오스 비드에 포함 된 정렬 된 바이러스 입자의 현미경을 스캔. 아가로 오스 비즈에서 SYBR 녹색 I-스테인드 바이러스 입자) 입체 재건. 삽입 : 분리 된 바이러스 입자의 높은 배율 아가로 오스 비즈에서 스테인드 하나의 바이러스 입자의 상대적 형광 B) 프로필 플롯.. <img alt="그림 3" src="/files/ftp_upload/3899/3899fig3.jpg" /> 그림 3. PCR을 사용하여 살균 식별.) &lt;/ strong&gt;를 멀티 플렉스 PCR 유전자형, 레인 T4 및 람다 특정 프라이머를 사용하여 : 1. TrackIt 1킬로바이트 플러스 사다리 (Invitrogen 사), 2. 람다 integrase (750 BP), 3. T4의 주요 캡시드 단백질 (1,050 BP), 4. 1. : 람다 integrase와 T4의 주요 캡시드 단백질의 혼합 B) 추가 유전자좌와 후속 람다 특정 PCR 더 파지 게놈 격리, 레인을 확인합니다. 람다 integrase (750 BP), 2. 람다 억제 (356 BP) 3. 람다 SIE (superinfection 제외) (456 BP) 4. TrackIt 1킬로바이트 플러스 사다리 (Invitrogen 사). <img alt="그림 4" src="/files/ftp_upload/3899/3899fig4.jpg" /> 그림 4. 람다 게놈 특성 및 적용.) GC 플롯, B) 람다의 게놈지도 (에서 적응 <a href="http://img.jgi.doe.gov" target="_blank">http://img.jgi.doe.gov</a> ) 및 C) SVG의 매핑 참조 박테리오파지 람다에 대한 읽기, x 축입니다 게놈 위치, 나중에XIS는 % 범위이다.

<ol>
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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. 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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>

관심 없음 충돌 선언하지 않습니다.

SVG 방법을 적용 할 때 중요한 요소의 수를 고려해야합니다. 유전자형은 개념 증명 실험 13에서 수행 한대로 보존 된 프라이머는 모든 바이러스 그룹에서 사용할 수없는 환경 적 또는 알 수 분리에 대한 올바른 옵션이 아닙니다. 또한, 배경 DNA 합성이나 비특이적 인 증폭은 일반적으로 MDA 반응 14를 사용하여 증폭하는 동안보고됩니다. 비특이적 증폭 반응 시약 및 / 나 반응 혼합물 ?...

<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'

단일 미생물 세포의 전체 게놈 증폭 및 염기 서열 재배 1-3의 필요없이 게놈 특성화 할 수 있습니다. 유비쿼터스와 우리의 행성 4에서 가장 많은 엔티티와 모든 환경 5 중요한 바이러스, 유사한 접근 방식을 통해 밝혀 질 못하고있다. 여기에서 우리는 &#39;단일 바이러스 게놈&#39;(SVG)라는 단일 비리의 게놈을 분리하고 특성화 방법을 설명합니다. SVG 후속 시퀀싱 반응에 사용할 수있는 고 분자량의 게놈 DNA (gDNA를)을 얻기 위해 각각의 바이러스 전체 게놈 증폭을 분리하는 유동 세포 계측법을 사용합니다.

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;

단일 바이러스 게놈 (SVG)는 단일 virons의 게놈을 분리하고 증폭하는 방법이다. 혼합 조립의 바이러스 현탁액하여 virion의 캡처 및 다운 스트림 처리하는 동안 게놈 전단을 줄이고, 아가로 오스를 포함하는 개별 우물 현미경 슬라이드에 유동 세포 계측법을 사용하여 정렬됩니다. 전체 게놈 증폭 시퀀싱에 적합한 유전 물질의 결과로 여러 변위 증폭 (MDA)를 사용하여 수행됩니다.

&#39;단일 바이러스 게놈&#39;을 사용하여 단일 비리의 분리 및 유전체 분석

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 조회수.  The J. Craig Venter Institute. 단일 바이러스 게놈 (SVG)는 단일 virons의 게놈을 분리하고 증폭하는 방법이다. 혼합 조립의 바이러스 현탁액하여 virion의 캡처 및 다운 스트림 처리하는 동안 게놈 전단을 줄이고, 아가로 오스를 포함하는 개별 우물 현미경 슬라이드에 유동 세포 계측법을 사용하여 정렬됩니다. 전체 게놈 증폭 시퀀싱에 적합한 유전 물질의 결과로 여러 변위 증폭 (MDA)를 사용하?...

비디오: 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.

에 관련된 호스트 요인에 대한 RNAi 심사 우두</em사용하기> 바이러스 감염 Drosophila 전지

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

연구

면역학 및 감염병학

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.

히드록 시아파 타이트 크로마 토그래피를 사용하여 환경 바이러스성 커뮤니티에서 단일 좌초 DNA, 더블 좌초된 DNA와 RNA의 분리

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.

의 단일 세포 분석 바실러스 subtilis</em형광 현미경 및 유동세포계측법 사용> Biofilms

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.

Photoactivated 단일 분자 추적을 사용하여 라이브 박테리아 세포에있는 단백질-DNA 상호 작용을 시각화

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.

다른 마우스 피부 지역에서 Cytofluorimetric 분석을위한 단일 세포 현탁액의 준비

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.

호중구의 분리 및 분석 치료제에 림프종 세포 감도에서 자신의 역할을 확인하는 방법

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.

madSTORM을 이용한 T 세포의 고해상도 이미징

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.

Immunophenotype 및 Cytokine 생산 대량 Cytometry 통해 주변 전 혈의 단일 세포 분석

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.

다운스트림 단일 세포 분석에 대 한 요로 감염의 Murine 모델에서 생성 된 단일 세포내 세균성 지역 사회의 절연

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.

단일 T 세포또는 면역 학적 단세포 힘 분광학을 위한 단입자를 가진 원자력 현미경 캔틸레버의 기능화

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