저자는이 기술을 개발하기위한 모든 EIB 회원 감사드립니다. 이 연구는 우선 지역 &quot;막 트래픽&quot;(호) 및 &quot;감염성 현상 매트릭스&quot;(KH), 젊은 과학자 (SF와 KH) 및 학술 연구 (호에 대한 과학 연구 보조금에 의해 부분적으로 지원되었다 혁신 분야 교육부, 문화, 스포츠, 과학 및 일본의 기술에서 &quot;세포내 물류&quot;(호)에), 그리고 과학 연구.

1. 세균성 세포의 준비 <ol><li> 줄무늬의 글리세롤는 LB에 (예 : GFP 표현 S. Typhimurium)에 형광 박테리아를 꽉찬 100 μg / 암피실린의 ML이 포함된 한천 플레이트. </li><li> 새로운 LB 매체 2 ML에서 하룻밤 LB 한천 배지에서 문화는 하나의 식민지. </li><li> 600 NM에서 1.0의 광학 밀도에 도달할 때까지 새로운 LB 매체와 부화 4.5 ML에 세균성 문화의 0.5 ML을 추가합니다. </li><li> 원심 분리하여 세포를 수확 세균 (3,000 XG, 5 분, 4 ° C). </li><li> 뜨는을 취소하고 멸균 인산 완충 식염수 (PBS)의 5.0 ML로 두 번 씻는다. </li><li> PBS 5 ML과 박테리아 펠렛을 Resuspend하고 inoculum로 약 10 7 콜로니 형성 단위 (CFU)이있는 정지 50 μl를 사용합니다. </li><li> 찬란 분류 박테리아를 사용하는 경우에는 세균성 세포는 표준 프로토콜에 따라 형광 라벨 시약에 의해 분류되었다. </li></ol> 2.nesthesia <ol><li> 5 % (V / V)와 함께 작은 플라스틱 상자 (10 × 10 × 5cm)를 기입하면 증발 isoflurane 공기 (유량 : 200 ML / 분)와 혼합.을 </li><li> 마취 여덟에있는 상자에 열여섯 - 약한 세의 남성 또는 여성 쥐. </li><li> 마취 후 부검 테이블에 마우스를 이동합니다. </li><li> 지속 2퍼센트 (V / V)에 의해 생쥐를 마취하는 것은 증발 isoflurane 공기 (유량 : 200 ML / 분)와 함께 혼합 (그림 1).를 이것은 터미널 절차입니다. </li></ol> 3. 출혈도 잡았 Peyer의 패치 루프 분석 <ol><li> 복부 피부와 다음의 절개 1cm가 anesthetized 마우스의 복부 복막을 잘라 Peyer의 패치를 포함하고있는 작은 창자를 꺼내. </li><li> 혈관을 피하기 위해 간병, 봉제 원사와 대장을 Ligate. 참고 : 대장만이 바인딩 한쪽, 그리고 느슨한 반대편 두십시오. </li><li> 느슨한의에서 출혈도 잡았 Peyer의 패치 루프에 세균 현탁액 또는 주사기로 PBS (제어) 50 μl를 주입대장의 IDE (그림 2). </li><li> 느슨한 측면을 바인딩, 그리고 클립과 마우스의 복부를 닫습니다. </li><li> 1 H 후, 출혈도 잡았 Peyer의 패치 루프를 제거하고 경추 전위에 의한 마우스를 안락사. </li><li> 출혈도 잡았 Peyer의 패치 루프에서 소비세 Peyer의 패치. </li><li> 초과 점막 액체와 세균을 제거 바늘에 연결된 주사기를 이용한 PBS의 1ml에 의해 Peyer의 패치의 혀끝의 측면을 번쩍. Peyer의 패치에게 추가로 두 번 깜박임. </li></ol> 4. 전체 마운트 얼룩과 Peyer의 패치 공촛점 현미경 분석 <ol><li> 1 시간을위한 얼음 BD Cytofix / Cytoperm 솔루션에 Peyer의 패치를 수정. </li><li> 30 분 0.1 % (W / V) 사포닌, 0.2 % (W / V) PBS에 BSA를 포함 차단 버퍼 1 ML과 블록 다음 BD 페름 / 5 분 버퍼를 세척 1ml와 Peyer의 패치 세 번 씻고, 그리고 얼음. </li><li> 200 배 희석이 방지 마우스를 표본으로 GP2 단클론 항체 (5 μg / ML)는 검색에 추가M 세포. </li><li> 4 2 실온에서 시간이나 야간을위한 품어 ° C. </li><li> 차가운 PBS 1 ML로 세 번 씻어 다음 표본에 DyLight549 (20 μg / ML)와 복합 200 배 희석 안티 쥐 IgG 항체를 추가합니다. </li><li> 2 시간을위한 얼음에 샘플을 품어. </li><li> 차가운 PBS 1 ML로 세 번 씻어 다음, F - 굴지을 감지하기 위해 시편 50 배 희석 알렉사 633 복합 Phalloidin를 추가합니다. </li><li> 2 시간을위한 얼음 알을 품다. </li><li> 차가운 PBS 1 ML로 가볍게 씻으십시오. 그런 다음 슬라이드에 원형 커버 유리 3-4 조각을 배치하고, 슬라이드 (그림 3)에서 PBS에서 글리세롤의 30 % 용액에 표본 포함. </li><li> DM - IRE2 공촛점 레이저 스캐닝 현미경이나 DeltaVision 복원 deconvolution 현미경과 표본을 관찰. </li></ol> 5. 대표 결과 : 마우스의 표본 예제는 GFP - S. 균과 Peyer의 패치 루프 분석 출혈도 잡았murium은 DeltaVision 복원 deconvolution 현미경 (그림 4)와 함께 관찰되었다. GFP - S. Typhimurium은 GP2 + M 세포에 의해 transcytosed 있습니다. 마우스 Peyer의 패치 장 루프 분석은 M 세포에 의해 transcytosed 수 있습니다 공생이나 병원성 세균의 종류를 식별할 수 있습니다. <img alt="그림 1" src="/files/ftp_upload/3225/3225fig1.jpg" /> 그림 1. 증발 isoflurane 조건하에 생쥐의 아나스타샤. 증발 isoflurane는 마취 - 전용 장치 (왼쪽)가 제공되었습니다. <img alt="그림 2" src="/files/ftp_upload/3225/3225fig2.jpg" /> 그림 2. 출혈도 잡았 Peyer의 패치 장 루프 분석의 요약. 형광 박테리아 정지는 대장의 느슨한 측면에 출혈도 잡았 Peyer의 패치 루프에 주사기로 주입했다. <img alt="그림 3" src="/files/ftp_upload/3225/3225fig3.jpg" /> <strong&gt; 그림 3. 표본의 마운트합니다. 표본은 구멍 플라스틱 플레이트는 (두께 1mm) 유리 슬라이드에 접착제 에이전트에 의해 묶여있는 특별한 슬라이드에 PBS에서 글리세롤의 30 % 용액에 포함되었습니다. <img alt="그림 4" src="/files/ftp_upload/3225/3225fig4.jpg" /> 그림 4. GFP - S. Typhimurium과 Peyer의 패치 루프 분석 출혈도 잡았 마우스의 예. M 세포 특정 마커 5보고 GP2는, 안티 - 마우스 GP2 항체 (적색)와 스테인드했습니다. 표본은 DeltaVision 복원 deconvolution의 현미경으로 관찰했다. GFP - 표현 S. Typhimurium은 M 세포의 꼭대기의 플라즈마 막과 subapical 세포질 vesicles (화살촉)에 GP2와 함께 공동화된되었습니다. 탑, 혀끝의 볼 수 있습니다. 하단과 측면, 측면 전망이 있습니다. 스케일 바 : 10 μm의.

<ol>
	<li>Fagarasan, S., Honjo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+IgA+synthesis:+regulation+of+front-line+body+defences.">Intestinal IgA synthesis: regulation of front-line body defences.</a> Nat. Rev. Immunol. 3, 63-72 (2003).</li><li>Hapfelmeier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+microbial+colonization+of+germ-free+mice+reveals+the+dynamics+of+IgA+immune+responses.">Reversible microbial colonization of germ-free mice reveals the dynamics of IgA immune responses.</a> Science. 328, 1705-1709 (2010).</li><li>Owen, R. L., Jones, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell+specialization+within+human+Peyer's+patches:+an+ultrastructural+study+of+intestinal+lymphoid+follicles.">Epithelial cell specialization within human Peyer's patches: an ultrastructural study of intestinal lymphoid follicles.</a> Gastroenterology. 66, 189-203 (1974).</li><li>Neutra, M. R., Mantis, N. J., Kraehenbuhl, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collaboration+of+epithelial+cells+with+organized+mucosal+lymphoid+tissues.">Collaboration of epithelial cells with organized mucosal lymphoid tissues.</a> Nat. Immunol. 2, 1004-1009 (2001).</li><li>Hase, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+through+glycoprotein+2+of+FimH(+)+bacteria+by+M+cells+initiates+mucosal+immune+response.">Uptake through glycoprotein 2 of FimH(+) bacteria by M cells initiates mucosal immune response.</a> Nature. 462, 226-230 (2009).</li><li>Punyashthiti, K., Finkelstein, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteropathogenicity+of+Escherichia+coli.+I.+Evaluation+of+mouse+intestinal+loops.">Enteropathogenicity of Escherichia coli. I. Evaluation of mouse intestinal loops.</a> Infect. Immun. 4, 473-478 (1971).</li><li>Owen, R. L., Pierce, N. F., Apple, R. T., Cray, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M+cell+transport+of+Vibrio+cholerae+from+the+intestinal+lumen+into+Peyer's+patches:+a+mechanism+for+antigen+sampling+and+for+microbial+transepithelial+migration.">M cell transport of Vibrio cholerae from the intestinal lumen into Peyer's patches: a mechanism for antigen sampling and for microbial transepithelial migration.</a> J. Infect. Dis. 153, 1108-1118 (1986).</li><li>Owen, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M+cells--entryways+of+opportunity+for+enteropathogens.">M cells--entryways of opportunity for enteropathogens.</a> J. Exp. Med. 180, 7-9 (1994).</li><li>Clark, M. A., Hirst, B. H., Jepson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M-cell+surface+beta1+integrin+expression+and+invasin-mediated+targeting+of+Yersinia+pseudotuberculosis+to+mouse+Peyer's+patch+M+cells.">M-cell surface beta1 integrin expression and invasin-mediated targeting of Yersinia pseudotuberculosis to mouse Peyer's patch M cells.</a> Infect. Immun. 66, 1237-1243 (1998).</li><li>Jensen, V. B., Harty, J. T., Jones, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+the+invasive+pathogens+Salmonella+typhimurium,+Listeria+monocytogenes,+and+Shigella+flexneri+with+M+cells+and+murine+Peyer's+patches.">Interactions of the invasive pathogens Salmonella typhimurium, Listeria monocytogenes, and Shigella flexneri with M cells and murine Peyer's patches.</a> Infect. Immun. 66, 3758-3766 (1998).</li><li>Nagai, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Peyer's+patches+in+the+induction+of+Helicobacter+pylori-induced+gastritis.">Role of Peyer's patches in the induction of Helicobacter pylori-induced gastritis.</a> Proc. Natl. Acad. Sci. U. S. A. 104, 8971-8976 (2007).</li></ol>

관심 없음 충돌 선언하지 않습니다.

<html> 세균 현탁액과 출혈도 잡았 Peyer의 패치의 부화 시간은 M 세포에 박테리아 정관을 준수하기 위해 1 시간을 위해 일반적이다. 4 시간 보육의 경우, 박테리아가 자주 Peyer의 패치의 T 세포 영역에 감지됩니다. 증발 isoflurane 흡입 마취는 마우스가 안정적으로 지킬 수로서, 세균 현탁액과 출혈도 잡았 Peyer의 패치의 배양 시간은 Peyer의 패치에 T - 세포 영역으로 이동 박테리아를 관찰 확장 가능합니다...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> LB 한천 암피실린 - 100 </td><td> 시그마 </td><td> L5667 </td><td></td></tr><tr><td> Cy3 모노 반응 염색약 </td><td> GE 헬스케어 </td><td> PA23001 </td><td></td></tr><tr><td> 알렉사 형석 350 카르복실산, succinimidyl 에스테르 </td><td> Invitrogen </td><td> A10168 </td><td></td></tr><tr><td> 흡입 마취 기구 </td><td> 시나 제작소 </td><td> SN - 487 </td><td></td></tr><tr><td> 고정 및 Permeabilization 용액 </td><td> BD </td><td> 554,722 </td><td></td></tr><tr><td> 안티 마우스 당단백질이 항독소 </td><td> MBL </td><td>D278 - 3 </td><td> 200 배 희석 (5μg/ml) </td></tr><tr><td> 염소 방지 쥐 IgG, F (AB &#39;) 2 조각 구체적인 </td><td> 잭슨 ImmunoResearch </td><td> 112-505-006 </td><td> 200 배 희석 (20μg/ml) </td></tr><tr><td> 알렉사 형석 633 Phalloidin </td><td> 분자 프로브 </td><td> A22284 </td><td> 50 배 희석 </td></tr><tr><td> 공촛점 레이저 현미경 스캐닝 </td><td> Leica 마이크로 시스템즈 </td><td> DMIRE2 </td><td></td></tr><tr><td> DeltaVision 복구 deconvolution 현미경 </td><td> 응용 정밀 </td><td> DeltaVision 코어 </td><td></td></tr></tbody></table>

application of a mouse ligated peyer&#x2019;s patch intestinal loop assay to evaluate bacterial uptake by m cells

우리의 창자의 안쪽은 공생 박테리아의 거대한 숫자 살고 있습니다. 위장관의 점막 표면은 지속적으로 그들과 때때로 병원균에 노출됩니다. 인내심을 관련 림프 조직 (갈트)는 이러한 미생물 1, 2에 대한 점막 면역 반응의 유도에 중요한 역할을한다. 점막 면역 반응을 시작하려면, 점막 항원이 같은 Peyer의 패치로 구성 림프 모공에 상피 장벽을 가로질러 내장 루멘에서 이송해야합니다. 이 항원 transcytosis는 전문 상피 세포 M 3, 4에 의해 중재됩니다. M 세포가 적극적으로 macromolecules과 미생물을 phagocytose 비정형 상피 세포 있습니다. 돌기 세포 (DCS)와 macrophages, 저하에 대한 lysosomes에 대상 항원과는 달리, M 세포는 주로 내면 항원을 transcytose. M 세포를 통해이 활발한 macromolecular의 transcytosis는 기본 구성 림프 모공에 항원을 제공합니다D는 항원에 특정한 점막 면역 반응을 시작을위한 필수적인 것으로 생각됩니다. 그러나, M 세포하여 항원 이해를 촉진 분자 메커니즘은 대부분 알 수 있습니다. 우리는 이전에 특별히 enterocytes 간의 M 세포의 꼭대기의 플라즈마 막에 표현 당단백질 2 (Gp2)는, 대장균과 살모넬라균 enterica serovar Typhimurium (S. Typhimurium을 포함하여 공생과 enterobacteria 병원성의 하위 집합에 대한 transcytotic 수용체로 역할을보고있다 ), FimH, 내가 세균성 외부 멤브레인 5 pili 유형의 구성 요소를 인식하여. 여기, 우리는 M 세포에 의해 박테리아 이해를 평가하기 위해 마우스 Peyer의 패치 장 루프 분석의 응용 프로그램에 대한 방법을 제시한다. 이 방법은 이전에 6, 7 장의 설명 마우스 루프 분석의 향상된 버전입니다. 개선된 점은 다음과 같습니다 : 1. Isoflurane은 마취제로 사용되었다. 2. 약 1cm 출혈도 잡았 장 루프 includING Peyer의 패치를 설치했습니다. 3. M 세포에 의해 촬영 박테리아는 찬란 형광 라벨 시약이나 녹색 형광 단백질 (GFP) 같은 형광 단백질 overexpressing로 분류되었습니다. 4. Peyer의 패치를 덮고 뿌리 - 관련 상피의 M 세포는 안티 Gp2 항체와 전체 마운트 immunostainig에 의해 발견되었다. 5. M 세포에 의해 형광 박테리아 transcytosis은 공촛점 현미경 분석에 의해 관찰되었다. 마우스 Peyer의 패치 장 루프 분석은 답변을 공급 수 어떤 종류의 공생 또는 M 세포에 의해 transcytosed 병원성 세균 및 M 세포를 통해 점막 면역 체계를 자극하는 방법의 분자 메커니즘을 이해로 우리를 이끌 수 있습니다.

Watch this Scientific Journal Video about Application of a Mouse Ligated Peyerâ€™s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells at JoVE.com

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

Peyer의 패치를 다루는 전문 뿌리 - 관련 상피의 M 세포는 창자 - 관련 림프 조직에서 점막 immunosurveillance을위한 중요한 역할을한다. 여기 우리는 M 세포에 의해 세균 transcytosis에 대한 평가 방법을 설명<em&gt; 생체내에</em&gt;. 이 방법은 면역 시스템에 M - 세포 기능을 이해하는 방법을 제공합니다.

마우스의 응용은 M 세포에 의해 박테리아 이해를 평가하는 Peyer의 패치 장 루프 분석 출혈도 잡았

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

application-mouse-ligated-peyers-patch-intestinal-loop-assay-to

Research

JoVE Journal

Immunology and Infection

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

19.4K 조회수.  RIKEN Research Center for Allergy and Immunology. Peyer의 패치를 다루는 전문 뿌리 - 관련 상피의 M 세포는 창자 - 관련 림프 조직에서 점막 immunosurveillance을위한 중요한 역할을한다. 여기 우리는 M 세포에 의해 세균 transcytosis에 대한 평가 방법을 설명 생체내에. 이 방법은 면역 시스템에 M - 세포 기능을 이해하는 방법을 제공합니다.

비디오: Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

마우스의 이미지 세균 감염에 루시페라제를 사용하여

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

연구

면역학 및 감염병학

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

세균성 병원체에 의해 인간의 세포의 침략

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

T6SS로 인한 세균 경쟁을 모니터링 할 비주얼 분석

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine via a series of lymphatic vessels and nodes that converge at the superior mesenteric lymph duct. Cannulation of the mesenteric lymph duct thus enables the collection of mesenteric lymph flowing from the intestine. Mesenteric lymph consists of a cellular fraction of immune cells (99% lymphocytes), aqueous fraction (fluid, peptides and proteins such as cytokines and gut hormones) and lipoprotein fraction (lipids, lipophilic molecules and apo-proteins). The mesenteric lymph duct cannulation model can therefore be used to measure the concentration and rate of transport of a range of factors from the intestine via the lymphatic system. Changes to these factors in response to different challenges (e.g., diets, antigens, drugs) and in disease (e.g., inflammatory bowel disease, HIV, diabetes) can also be determined. An area of expanding interest is the role of lymphatic transport in the absorption of orally administered lipophilic drugs and prodrugs that associate with intestinal lipid absorption pathways. Here we describe, in detail, a mesenteric lymph duct cannulated rat model which enables evaluation of the rate and extent of lipid and drug transport via the lymphatic system for several hours following intestinal delivery. The method is easily adaptable to the measurement of other parameters in lymph. We provide detailed descriptions of the difficulties that may be encountered when establishing this complex surgical method, as well as representative data from failed and successful experiments to provide instruction on how to confirm experimental success and interpret the data obtained.

Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

장간막 림프 덕트 유관 쥐 모델 : 창자 림프 약물 운송의 평가에 응용

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Application of Long-term cultured Interferon-&amp;#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

소에 이펙터 및 메모리 T 세포 반응을 평가하기위한 장기 배양 인터페론-γ 효소 링크 된 Immunospot 분석의 응용 프로그램

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

단핵구 단층 분석의 사용은 Fcγ 수용체 - 매개 식균 작용을 평가하기

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of infected cells, including organelle ultrastructure, cytoskeletal network organization, as well as cellular processes such as Stress Granule (SG) formation. The characterization of how pathogens subvert host processes is an important and integral part of the field of pathogenesis. While variable phenotypes may be readily visible, the precise analysis of the qualitative and quantitative differences in the cellular structures induced by pathogen challenge is essential for defining statistically significant differences between experimental and control samples. SG formation is an evolutionarily conserved stress response that leads to antiviral responses and has long been investigated using viral infections1. SG formation also affects signaling cascades and may have other still unknown consequences2. The characterization of this stress response to pathogens other than viruses, such as bacterial pathogens, is currently an emerging area of research3. For now, quantitative and qualitative analysis of SG formation is not yet routinely used, even in the viral systems. Here we describe a simple method for inducing and characterizing SG formation in uninfected cells and in cells infected with a cytosolic bacterial pathogen, which affects the formation of SGs in response to various exogenous stresses. Analysis of SG formation and composition is achieved by using a number of different SG markers and the spot detector plug-in of ICY, an open source image analysis tool.

We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions.

포유류 세포의 박테리아 감염 후 스트레스 과립 형성의 면역 형광 분석

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

상처 치유 분석 결과 셀 마이그레이션 평가 하는 새로운 체 외에

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

Cell Membrane Repair Assay Using a Two-photon Laser Microscope

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can result in disease. Understanding the underlying molecular mechanisms surrounding cell membrane repair is, therefore, an important objective to the development of novel therapeutic strategies for diseases associated with dysfunctional cell membrane dynamics. Many in vitro and in vivo studies aimed at understanding cell membrane resealing in various disease contexts utilize two-photon laser ablation as a standard for determining functional outcomes following experimental treatments. In this assay, cell membranes are subjected to wounding with a two-photon laser, which causes the cell membrane to rupture and fluorescent dye to infiltrate the cell. The intensity of fluorescence within the cell can then be monitored to quantify the cell's ability to reseal itself. There are several alternative methods for assessing cell membrane response to injury, as well as great variation in the two-photon laser wounding approach itself, therefore, a single, unified model of cell wounding would beneficially serve to decrease the variation between these methodologies. In this article, we outline a simple two-photon laser wounding protocol for assessing cell membrane repair in vitro in both healthy and dysferlinopathy patient fibroblast cells transfected with or without a full-length dysferlin plasmid.

Cell membrane wounding via two-photon laser is a widely used method for assessing membrane resealing ability and can be applied to multiple cell types. Here, we describe a protocol for in vitro live-imaging of membrane resealing in dysferlinopathy patient cells following two-photon laser ablation.

세포 막 복구 분석 결과 2 광자 레이저 현미경을 사용 하 여

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can ...

Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water environment. Developing effective methods for oral delivery of immunostimulants or vaccines, which activate the immune system against infectious diseases, is highly desirable. In devising prophylactic tools, good experimental models are needed to test their performance. Here, we show a method for oral intubation of adult zebrafish and a set of procedures to dissect and prepare the intestine for cytometry, confocal microscopy and quantitative polymerase chain reaction (qPCR) analysis. With this protocol, we can precisely administer volumes up to 50 &#181;L to fish weighing approximately 1 g simply and quickly, without harming the animals. This method allows us to explore the direct in vivo uptake of fluorescently labelled compounds by the intestinal mucosa and the immunomodulatory capacity of such biologics at the local site after intubation. By combining downstream methods such as flow cytometry, histology, qPCR and confocal microscopy of the intestinal tissue, we can understand how immunostimulants or vaccines are able to cross the intestinal mucosal barriers, pass through the lamina propria, and reach the muscle, exerting an effect on the intestinal mucosal immune system. The model could be used to test candidate oral prophylactics and delivery systems or the local effect of any orally administered bioactive compound.

The protocol describes intubating adult zebrafish with a biologic; then dissecting and preparing the intestine for cytometry, confocal microscopy and qPCR. This method allows administration of bioactive compounds to monitor intestinal uptake and the local immune stimulus evoked. It&#160;is relevant for testing the intestinal dynamics of oral prophylactics.