The authors thank Ian Clarke and P. Scott Hefty for the pGFP-SW2 and pASK-GFP-mKate2-L2 plasmids, respectively. We thank Paul Miller for technical assistance and Richard Stephens for other resources. This work was funded by NIH grant AI095603 (KH).

형광 숙주 세포 라인의 1 세대 <ol><li> / 잘 2 × 10 6 세포에서 6 웰 플레이트에 일 1. 플레이트 293T 세포 (또는 다른 레트로 바이러스 포장 라인) ~ 75 % 합류 다음날합니다. 원한다면, 각각의 레트로 바이러스에 대한 중복 웰 플레이트가 사용될 수있다. </li><li> 주 2. 대기음 세포 및 2를 가하여 신선한 성장 배지 (DMEM + 10 % FBS + 2 mM의 L- 글루타민). 리포 펙 타민 2000 또는 유사한 시약을 사용하여 제조업체의 지침에 따라 5-8 μg의 레트로 바이러스 벡터 (GFP를 포함) 및 포장 벡터의 각 (예., pVSVg)와 세포를 형질. </li><li> 1 ml의 성장 매체와 매체를 교체 이후 37 ° C CO 2 배양기에서 4-6 시간 동안 DNA와 세포를 품어합니다. </li><li> 37 ° C CO 2 배양기에서 48 시간 동안 세포를 품어. </li><li> 일 3. 플레이트 표적 세포 (예., 헬라) 105 세포의 밀도로 대략 6 웰 플레이트 상 / 웰이 될 ~ 50 % 합류다음날. 거꾸로 현미경에 GFP의 형광을 모니터링하여 293T 세포에 긍정적 인 형질을 확인합니다. </li><li> 주 4. 수집 레트로 바이러스 함유 0.45 μm의 셀룰로오스 아세테이트 주사기 필터를 통해 뜨는을 피펫을 사용하여 필터링 293T 세포 오프 상층 액을. </li><li> 헬라 세포에서 매체를 제거하고 16 μg의 / ㎖의 폴리 브렌를 포함하는 0.5 ml의 성장 매체를 추가합니다. 세포에 필터링 된 레트로 바이러스의 ~ 0.5 ML을 추가 현명한 드롭, 24 시간 동안 37 ° C CO 2 배양기에서 세포를 배양한다. 4 ℃에서 (잘 복제에서, 즉) 남아있는 레트로 바이러스를 저장합니다. </li><li> 중복 우물이 순차적으로 나머지 레트로 바이러스 입자와 감염 여분의 레트로 바이러스, 단계를 반복 1.7을 생성하는 사항이있는 경우의 날 (5). </li><li> (예를 들어, 500 μg의 / ㎖ 네오 마이신) 10cm 접시 포함 된 선택 항생제로 1 일 6 항로는 헬라 세포 1을 형질 전환. </li><li> 선택 antibio의 존재에 다시 성장 형질 세포에 충분한 시간을 기다립니다셀 선택 항생제의 낮은 농도의 표준 기술에 의해 전달 될 ​​수있는 후 TIC (예., 200 μg의 / ㎖ 네오 마이신). 주 : 필요한 경우 세포 클론 적 또한,이 때 이상적인 밝기 위해 선택 될 수있다. </li></ol> GFP 발현 클라미디아 트라코마 티스의 2 세대 <ol><li> 배 CaCl2를 버퍼 (20 mM 트리스 산도 7.4, 100 mM의 염화칼슘 2)와 4SP 버퍼 (0.4 M 자당, 16 mM의 나 2 HPO 4)를 준비합니다. </li><li> 주 1. 해동 냉동 C. 트라코마 티스 재고 얼음에 즉시 50 μL 배 CaCl2를 버퍼에 10 μL 박테리아를 희석. 3 μg의 플라스미드 DNA를 추가 (예., PASK-GFP - mKate2-L2)를 잘 혼합하고 25 ℃에서 30 분 동안 품어. </li><li> 이 단계 동안, 원심 분리 튜브에 맥코이 세포를 T-75 플라스크를 trypsinizing하여 숙주 세포를 준비한다. 5 분 50 XG에 세포 현탁액을 원심 분리기 및 상층 액을 버린다. Resuspen1X CaCl2를 버퍼의 적절한 체적 D 세포 펠렛을 4 × 10 7 세포 / ㎖의 최종 농도를 얻었다. </li><li> (단계 2.2)에서 30 분간 인큐베이션 한 후, 형질 전환 혼합물을 튜브 (200 μl의 총 부피)에 맥코이 세포를 제조 100 μL를 추가하고, 25 ℃에서 추가로 20 분 동안 배양한다. 2 ml의 성장 매체와 6 자 플레이트와 오버레이에서 하나의 잘이 형질 전환 된 세포 혼합물의 100 μl를 추가합니다. 2 일간 CO 2 인큐베이터에서 37 ℃에서 배양한다. </li><li> 일 3.를 Lyse 맥코이 세포는 C. 감염 구부러진 1,000 μL 피펫 팁으로 세포를 1 ML의 DH 2 O와 매체를 대체하고 긁어 트라코마 티스. C의 효율적인 세포 용해 및 릴리스 5 회 1 ml의 4SP 버퍼를 추가하고 ~ 27.5 G 바늘을 통해 서스펜션을 통과 트라코마 티스. </li><li> C.를 함유하는 세포 용 해물의 전사 2 ml를 갓 도금 MCC의 합류 단층을 함유하는 T-75 플라스크에 트라코마오우 세포; (FBS)없이 8 mL의 DMEM을 추가 C. 있도록 25 ℃에서 2 시간 동안 배양 트라코마는 세포에 부착합니다. </li><li> 기음 세포는 10 U / ml의 페니실린 G와 1 μg의 / ㎖의 사이클로 헥시 미드와 보충 신선한 성장 매체를 추가하고. 37 ° C CO 2 배양기에서 48 시간 동안 플라스크를 품어. </li><li> 일 4. 세포가 감염되고 흠이 비정상적 클라미디아를 포함하는 광학 현미경으로 확인합니다. 2보다 큰 μm의 직경이다 흠 내 링 형상의 객체로 표준 광학 현미경에 밝은 공포, 그리고 비정상적인 클라미디아 기관 등의 흠을 확인합니다. </li><li> 현탁액으로 세포를 2 ML의 DH 2 O와 매체를 대체하고 긁어로 날 (5)를 Lyse 문화. 2 ml의 4SP 버퍼를 추가하고 ~ 27.5 G 바늘을 통해 5 번 정지를 전달합니다. </li><li> 6 웰 플레이트에 하나의 웰에 맥코이 세포의 신선한 단층을 감염 세포 용 해물을 2 ㎖를 사용합니다. 25 ° C, 흡인에서 2 시간 동안 해물과 세포를 품어 신선한 성장을 추가페니실린 G 및 사이클로 헥시 미드를 포함하는 매체. </li><li> 세포의 건강에 따라 3 ~ 5 일 동안 37 ° C CO 2 배양기에서 세포를 품어. </li><li> 반복 (비정상적인 몸의 결여) 정상 찾고 흠이 등장 할 때까지 6 웰 플레이트에 신선한 우물에 2.9-2.10 한 번 이상, 필요에 따라, 계대 배양 단계를 반복합니다. 이 때 확인하기 위해 거꾸로 형광 현미경을 사용 C. 트라코마는 mKate2 (빨간색)을 표현한다. </li><li> T-150 플라스크에서 재배 감염 맥코이이나 헬라 세포에서 클라미디아의 형질 전환을 수확하여, 예를 들어 높은 역가 클라미디아 주식의 생성에 감염된 문화, 최대 확장 할 수 있습니다. </li></ol> 3. 클라미디아와 세포를 감염 <ol><li> IFU 결정 및 멀티 웰 검사 예를 들어 유리 바닥 요리 또는 챔버 슬라이드 고해상도 이미징을위한, 또는 24 웰 플레이트 원하는 배양 용기에 플레이트 GFP - 헬라 세포. </li><li> 이리저리 해동C를 포함하는 선 튜브 트라코마 티스, C. muridarum, 또는 C 예를 MOI 1 = 얼음에 폐렴 및 감염의 원하는 다양성 (MOI)에 HBSS에 희석. </li><li> 사용되는 특정 클라미디아의 변형을 기반으로 정적 또는 원심 분리 주었 중 감염을 수행합니다. <ol><li> C.와 감염 트라코마 티스 LGV의 혈청 형 (L2) 또는 C muridarum, HBSS로 GFP - 헬라 세포를 세척하고 25 ℃에서 2 시간 동안 희석 세균 배양한다. 기음과는 HBSS로 세포를 씻어, 37 ° C CO 2 배양기에서 배양 배지에서 세포를 배양한다. </li><li> C. trachomatis에의 혈청 형의 D 또는 C와 감염 폐렴은, HBSS로 GFP - 헬라 세포를 씻어 세포에 희석 박테리아를 추가 할 수 있습니다. 벤치 탑 원심 분리기에서 스윙 버킷 회 전자 첨부의 멀티 웰 플레이트 홀더 멀티 웰 플레이트 (멀티 웰 플레이트 뚜껑을 테이프로 고정) 유리 바닥 접시를 놓습니다. </li><li> 1 시간 동안 25 ° C에서 900 XG에 원심 분리기 세포. </lI&gt; <li> 1 시간 동안 37 ° C CO 2 배양기에서 원심과 장소에서 문화 혈관을 제거합니다. </li><li> 기음 세포는 바이오 안전성 캐비닛에, HBSS로 두 번 세포를 씻어 신선한 성장 매체를 추가합니다. 37 ° C CO 2 인큐베이터에 넣어 세포. </li></ol></li></ol> 클라미디아 흠 4. 라이브 셀 시각화 <ol><li> CO 2 배양기에서 클라미디아 감염 GFP - 헬라 세포를 제거하고 페놀 5 + 빨간색 %의 FBS없이 RPMI와 매체를 교체하십시오. </li><li> 20 배 이상 오일 목표를 사용하여 거꾸로 형광 현미경 (니콘 이클립스 티-E 또는 이와 유사한 것)에 마운트 세포. </li><li> 큰 블랙홀 (클라미디아 흠)를 포함하는 녹색 세포 : 이미징 소프트웨어 (Volocity 6 또는 유사)를 사용하여,에 초점을 식별 클라미디아는 GFP - 헬라 세포를 감염. </li><li> 마크 이미지 소프트웨어를 이용하여 관심있는 클라미디아 -infected 세포를 포함하는 영역의 XYZ 위치 (용적ocity 6 또는 이와 유사한 것). 수동으로 XY 스테이지보기 모드에서 &#39;포인트 추가&#39;를하여이 작업을 수행합니다. 이러한 방식으로, XYZ 좌표는 각 표시된 위치에 저장됩니다. </li><li> 취득 설치 대화 상자에서, 녹색 (GFP, 헬라 세포질)과 빨간색 (mKate2, C. trachomatis에) 채널, 채널마다 새로운 프레임마다 5 분의 속도를 얻기 위해 소프트웨어를 구성합니다. </li><li> 프로토콜이 &quot;캡처 / 녹음&quot;버튼을 클릭하여 상기 입력을 이용하여 획득 된 이미지 시퀀스를 획득. </li></ol> 라이브 세포에 포함 성장 매개 변수 5. 정량 <ol><li> 원하는 시간에 후 감염 (예., 24, 48 HPI) 클라미디아는 인큐베이터에서 GFP - 헬라 세포를 감염 제거하고 거꾸로 형광 현미경에 장착합니다. 참고 : 높은 NA와 20 배 또는 40 배 공기의 목표는 24 웰 플레이트에 가장 적합하고, 40X 오일 목적은 챔버 슬라이드를 사용하는 것이 좋습니다. 이 엉덩이에 대한 중 하나를 기판에 세포를 성장AY. </li><li> 흠이 자신의 최대 직경에있는 세포의 미드 플레인에 초점 선명한 가장자리를 얻을 수 있습니다. </li><li> 각 웰 (물론 당 적어도 10)에 많은 분야에 대한 이미지를 수집; 우물은 일반적으로 복제 또는 실험 변수를 나타냅니다. </li><li> 눈으로 (개재물 GFP-HeLa 세포 검정 구획이다), 수동 개재물의 개수를 열거 나 자동으로 개재물을 식별하고 카운트하기 위해 이미지 소프트웨어 알고리즘을 사용한다. <ol><li> 형광 강도에 의해 GFP - 헬라 세포를 찾아 (명령 &#39;개체 찾기&#39;)를 세포의 상대 강도를 기반으로 형광 임계 강도를 조절합니다. </li><li> 그 단지보다 5 μm의 2를 유지의 크기 지침을 사용하여 필터 선택한 객체. 이는 &#39;인구 1&#39;이다. </li><li> 입력으로 &#39;인구 1&#39;을 사용하여 명령 &#39;개체에 구멍 채우기&#39;를 적용합니다. 이 단계는 &quot;인구 2 &#39;를 생성한다. </li><li> &#39;페이지에서 빼기&#39;인구 1 &#39;opulation 2 &#39;인구 3&#39;는 긍정적으로 지정 클라미디아 흠을 표시 얻었다 &#39;. </li><li> 예를 유지하는 객체에 대해, &#39;인구 3&#39;( &#39;필터 인구&#39;명령)에 크기 필터를 적용 24 시간 이상 감염된 세포에서 흠보다 큰 25 μm의 2. 이 흠이 훨씬 작 등의 이전에 18 시간 일찍 감염 시간, 내용, 집합 크기 필터는 4 μm의 2 이상의 개체를 유지합니다. &#39;흠​​&#39;로 지정된 객체의 인구 크기 필터링 결과. </li><li> 이미징 소프트웨어를 사용하여 정량 예컨대 이미지 데이터, 포접 번호, 개재물 크기 및 포접 둘레를 계산한다. </li></ol></li></ol>

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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=Chlamydia+pneumoniae--an+infectious+risk+factor+for+atherosclerosis.">Chlamydia pneumoniae--an infectious risk factor for atherosclerosis.</a> Nature Reviews Microbiology. 2 (1), 23-32 (2004).</li><li>Darville, T., Hiltke, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+genital+tract+disease+due+to+Chlamydia+trachomatis.">Pathogenesis of genital tract disease due to Chlamydia trachomatis.</a> The Journal of Infectious Diseases. 201, S114-S125 (2010).</li><li>Hackstadt, T., Stephens, 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=Cell+biology.">Cell biology.</a> Chlamydia: Intracellular Biology, Pathogenesis, and Immunity. , 101-138 (1999).</li><li>Wang, Y., Kahane, S., Cutcliffe, L. T., Skilton, R. J., Lambden, P. R., Clarke, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+transformation+system+for+Chlamydia+trachomatis:+restoration+of+glycogen+biosynthesis+by+acquisition+of+a+plasmid+shuttle+vector).">Development of a transformation system for Chlamydia trachomatis: restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector).</a> PLoS Pathogens. 7 (9), e1002258 (2011).</li><li>Hackstadt, T., Scidmore, M. A., Rockey, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+metabolism+in+Chlamydia+trachomatis-infected+cells:+directed+trafficking+of+Golgi-derived+sphingolipids+to+the+chlamydial+inclusion.">Lipid metabolism in Chlamydia trachomatis-infected cells: directed trafficking of Golgi-derived sphingolipids to the chlamydial inclusion.</a> Proceedings of the National Academy of Sciences of the United States of America. 92 (11), 4877-4881 (1995).</li><li>Boleti, H., Ojcius, D. M., Dautry-Varsat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+labelling+of+intracellular+bacteria+in+living+host+cells.">Fluorescent labelling of intracellular bacteria in living host cells.</a> Journal of Microbiological Methods. 40 (3), 265-274 (2000).</li><li>Hybiske, K., Stephens, 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=Mechanisms+of+host+cell+exit+by+the+intracellular+bacterium+Chlamydia.">Mechanisms of host cell exit by the intracellular bacterium Chlamydia.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (27), 11430-11435 (2007).</li><li>Heinzen, R. A., Hackstadt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Chlamydia+trachomatis+parasitophorous+vacuolar+membrane+is+not+passively+permeable+to+low-molecular-weight+compounds.">The Chlamydia trachomatis parasitophorous vacuolar membrane is not passively permeable to low-molecular-weight compounds.</a> Infection and Immunity. 65 (3), 1088-1094 (1997).</li><li>Wickstrum, J., Sammons, L. R., Restivo, K. N., Hefty, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+gene+expression+in+Chlamydia+trachomatis+using+the+tet+system.">Conditional gene expression in Chlamydia trachomatis using the tet system.</a> PLoS One. 8 (10), 10-1371 (2013).</li><li>Chin, E., Kirker, K., Zuck, M., James, G., Hybiske, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+recruitment+to+the+Chlamydia+inclusion+is+spatiotemporally+regulated+by+a+mechanism+that+requires+host+and+bacterial+factors.">Actin recruitment to the Chlamydia inclusion is spatiotemporally regulated by a mechanism that requires host and bacterial factors.</a> PLoS One. 7 (10), e46949 (2012).</li><li>Agaisse, H., Derr&#233;, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+C.+trachomatis+cloning+vector+and+the+generation+of+C.+trachomatis+strains+expressing+fluorescent+proteins+under+the+control+of+a+C.+trachomatis+promoter.">A C. trachomatis cloning vector and the generation of C. trachomatis strains expressing fluorescent proteins under the control of a C. trachomatis promoter.</a> PLoS One. 8 (2), e57090 (2013).</li></ol>

저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

여기에서 우리는 실시간으로 시각화 및 클라미디아 흠의 분석을위한 형광 숙주 세포를 생성하는 실험적인 전략을 설명합니다. 이 공포의 시각화 방법은 시간이 지남에 따라 단일 세포 세포의 인구를 통해 또는에서 클라미디아 개재물의 동적 특성을 조명 추적하고 정량적으로 측정 할 수있는 강력한 능력을 부여한다. 클라미디아 흠을 형광 단백질 표지 세포에서 눈에 띄게 잘 그들?...

세포 내 세균의 클라미디아의 종에 의해 발생하는 감염성 질환은 성병, 골반 염증성 질환, 실명, 폐렴 가능성이 동맥 경화 1-4을 포함하여 세계 보건에 큰 부담을 유도. 액포 내에서, 숙주 세포와 상호 작용하는 클라미디아의 능력 (포함 되나), 숙주 세포의 성공적인 감염 중요한 결정 요인이다. 포함은 클라미디아 성장을 가능하게하고 동적으로 클라미디아 5의 전체 2~3일 발달주기에 걸쳐 수정 된 새로운 병원성 구획입니다. chlamydiae의 의무적 세포 내 성격은 직접 포함의 독특한 생물학을 연구, 특히 연구 커뮤니티에 많은 과제를 제시한다. 주요 장애는 효율적으로 세포 내 클라미디아 또는 형광 접근하여 그들의 포함를 시각화하는 무능력하고있다살아있는 세포에서 말이지. 최근의 발견은 마지막으로 GFP C를 표현을 생성하는 방법을 공개했다 트라코마 티스 (6); 그러나,이 연구 결과는 아직 포함 특정 라벨을 주도하지 않았습니다. 일부 기술은 박테리아 나 개재물 7,8의 표시에 대해 기재되었지만, 이러한 photobleaching에 비 - 특이 조로 감수성과 같은 단점을 겪는다. 우리 그룹에 의해 중요한 발견은 숙주 세포 (9)을 표현 GFP를 사용하여 포함을 조명하기위한 새로운 전략을 수립. 이 전략은 합리적 이상 520 다 10 분자 포접 막의 극한 투과성을 이용한다. 세포가 안정적으로 특정 세포질 형광 단백질 (예를 들면, GFP 또는 mCherry)를 발현하도록 유전자 조작 된 경우, 클라미디아 흠 형광 자신의 완전한 배제로 놀라운 선명도와 함께 볼 수 있습니다. 이 역 이미징 전략은 모든 엽록소에 대한 흠 즉시 시각화 할 수 있습니다amydia 종과 용이 관심 대부분의 숙주 세포에 대해 적응 될 수있다. 그 유틸리티의 시연으로,이 방법은 클라미디아 종 9 휴대 출구 경로를 공개하고 정의하기 위해 이전에 사용되었다. 여기서, 우리는 또한이 방법을 수행하고, 개재물의 성장 역학에 관한 정량적 데이터 키를 유도하기 위하여 이용 될 수 있는지 보여준다. 또한, 비용 효율적 항체 기반 열거 방법을 대체 할 수 있으며 클라미디아 11 mKate2 발현과 같은 다른 형광 라벨과 협력하여 사용될 수있다. 공구의 이러한 강력한 결합은 살아있는 숙주 세포 내부 클라미디아 포함 막의 물성을 탐사 할 수있다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-Mem</td>
			<td>Invitrogen</td>
			<td>31985</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Sigma</td>
			<td>H9268</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 &#181;m filters</td>
			<td>Fisher</td>
			<td>09-719D</td>
			<td></td>
		</tr>
		<tr>
			<td>G418</td>
			<td>Invitrogen</td>
			<td>10131</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Invitrogen</td>
			<td>14025</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>11995</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Invitrogen</td>
			<td>11875</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 w/o phenol red</td>
			<td>Cellgro</td>
			<td>17-105-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin G</td>
			<td>Sigma</td>
			<td>13752</td>
			<td></td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma</td>
			<td>C7698</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom dishes</td>
			<td>MatTek</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Chamber slides, Lab-Tek II</td>
			<td>Nunc</td>
			<td>154526, 154534</td>
			<td></td>
		</tr>
	</tbody>
</table>

using fluorescent proteins to visualize and quantitate chlamydia vacuole growth dynamics in living cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

세포질 형광 단백질 (예를 들면, GFP)를 발현하는 포유 동물 세포는 살아있는, 감염된 세포 배양에서 클라미디아 흠의 조명을 사용하도록 설계 될 수있다. 클라미디아 감염시, 포함은 숙주 세포 (그림 1)에 검은 반점으로 쉽게 볼 수 있습니다. 형광 부족 개재물의 선명도는보기 및 / 또는 치료 (그림 1)의 여러 분야에 걸쳐 개재물의 자동 식별을 위해 이용...

Watch this Scientific Journal Video about Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells at JoVE.com

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

시각화 및 정량하기 위해 형광 단백질을 사용하여<em&gt; 클라미디아</em&gt; 액포 성장 역학 살아있는 세포에서

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

using-fluorescent-proteins-to-visualize-quantitate-chlamydia-vacuole

Research

JoVE Journal

Immunology and Infection

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

7.5K 조회수.  University of Washington. A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

비디오: Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

유동세포계측법로 고정하고 살아있는 세포에서 Calpain 활동을 측정

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

연구

면역학 및 감염병학

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

셀을 별 모양으로 일곱 단계

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

에 셀 운명을 따라<em&gt; E. 대장균</em&gt; 파지 람다에 의해 감염 후

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. 
We generated model xenograft tumors in mice by subcutaneous injection of a human ovarian cancer cell line, OVCAR-8 (NCI DCTD Tumor Repository, Frederick, MD). 
We transformed attenuated strains of S. typhimurium bacteria (ELH430:SL1344 phoPQ- 1) with a constitutively expressed luciferase (luxCDABE) plasmid for visualization2. These strains specifically colonize tumors while remaining essentially non-virulent to the mouse1. 
Once measurable tumors were established, bacteria were injected intravenously via the tail vein with varying dosage. Tumor-localized, bacterial gene expression was monitored in real time over the course of 60 hours using an in vivo imaging system (IVIS). At each time point, tumors were excised, homogenized, and plated to quantitate bacterial colonies for correlation with gene expression data. 
Together, this data yields a quantitative measure of the in vivo growth and gene expression dynamics of bacteria growing inside tumors.

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. This video covers tumor cell preparation and implantation, bacteria preparation and injection, whole-animal luminescence imaging, tumor excision, and bacterial colony counting.

종양 표적의 측정 성장과 유전자 발현 역학<em&gt; S. 티피 뮤 리움</em&gt; 박테리아

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium ...

Forward Genetic Approaches in Chlamydia trachomatis

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its intractability to experimental transformation with recombinant DNA. We developed an approach to perform genetic analysis in C. trachomatis&#160;despite the lack of molecular genetic tools. Our method involves: i.) chemical mutagenesis to rapidly generate comprehensive libraries of genetically-defined mutants with distinct phenotypes; ii.) whole-genome sequencing (WGS) to map the underlying genetic lesions and to find associations between mutated gene(s) and a common phenotype; iii.) generation of recombinant strains through co-infection of mammalian cells with mutant and wild type bacteria. Accordingly, we were able to establish causal relationships between genotypes and phenotypes. The coupling of chemically-induced gene variation and WGS to establish correlative genotype&#8211;phenotype associations should be broadly applicable to the large list of medically and environmentally important microorganisms currently intractable to genetic analysis.

Forward Genetic Approaches in Chlamydia trachomatis

We describe a methodology to perform genetic analysis in Chlamydia based on chemical mutagenesis and whole genome sequencing. In addition, a system for DNA exchange within infected cells is described that can be used for genetic mapping. This method may be broadly applicable to microbial systems lacking transformation systems and molecular genetic tools.

앞으로 유전 접근<em&gt; 클라미디아의 trachomatis</em

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

막 극성, 멤브레인 무결성 및 세포 내 이온 농도의 모니터링 변경<em&gt; 폐렴 구균</em&gt; 형광 염료를 사용

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

형광 단백질을 사용하면 아프리카 Trypanosome에서 Glycosome 역학을 모니터링하려면

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

&quot;Phagosome Closure Assay&quot; to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

&quot;Phagosome 폐쇄 분석은&quot;사용하여 입체적으로 Phagosome 형성을 시각화하는 총 내부 반사 형광 현미경 (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

정밀 절단 라이브 폐 수지상 세포를 시각화하기 위해 마우스 폐 슬라이스

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.