Name: immunofluorescence analysis of stress granule formation after bacterial challenge of mammalian cells
Uploaded: 2017-07-03T13:00:00.000+00:00
Duration: 11 min 37 s
Description: Watch this Scientific Journal Video about Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells at JoVE.com

PS는 Bill and Melinda Gates 그랜드 챌린지 그랜트 OPP1141322의 수령자입니다. PV는 스위스 국립 과학 재단 Early Postdoc Mobility 펠로우쉽과 Roux-Cantarini 박사후 연구원의 후원으로 지원되었습니다. PJS는 HHMI 보조금 및 ERC-2013-ADG 339579-Decrypt에서 지원됩니다.

<ol>
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저자는 공개 할 것이 없습니다.

여기에 설명 된 프로토콜은 외인성 스트레스의 존재 또는 부재 하에서 cytosolic 병원체 S. flexneri 에 감염된 세포 및 감염되지 않은 세포에서 SG의 유도, 국소화 및 분석을 설명합니다. 자유 이미징 소프트웨어를 사용하여 프로토콜은 주어진 phenotypes의 차이를 식별하고 통계적으로 처리하기 위해 SG 형성에 대한 정확한 질적 및 양적 분석을 허용합니다. 감염, SG 유도 및 영...

세포 수준에서 숙주 - 병원체 상호 작용을 시각화하는 것은 병원성 전략에 대한 통찰력을 얻고 주요 세포 경로를 확인하는 강력한 방법입니다. 사실 병원균은 중요한 세포 표적이나 구조를 정확히 찾아내는 도구로 사용될 수 있습니다. 병원균은 생존이나 번식을위한 전략으로 중앙 세포 과정을 파괴하기 위해 진화했습니다. 형광 성분을 가진 숙주 단백질을 재조합 적으로 발현시킴으로써 세포 성분의 시각화를 달성 할 수 있습니다. 이것은 실시간 분석을 가능하게하지만, 특이 적으로 표지 된 숙주 단백질을 갖는 세포주의 생성은 매우 힘들고 바람직하지 않은 부작용을 초래할 수있다. 다중 항원 인자를 동시에 분석 할 수 있고 특정 세포 유형에만 국한되지 않기 때문에 특정 항체를 사용하여 세포 인자를 검출하는 것이 더 편리합니다. 단점은 immunofluorescence 분석은 숙주 세포 고정을 필요로하기 때문에 고정보기 만 캡처 할 수 있다는 것입니다이온. 그러나, immunofluorescence 이미징의 중요한 장점은 쉽게 질적 및 양적 분석 모두에 도움이된다는 것입니다. 이것은 차례로 호스트 - 병원체 상호 작용에 대한 새로운 통찰력을 제공하기 위해 통계적으로 중요한 차이를 얻는 데 사용될 수 있습니다. 형광 이미지 분석 프로그램은 3D 및 4D 분석을 수행하는 강력한 분석 도구입니다. 그러나 소프트웨어 및 유지 보수 비용이 높기 때문에 무료 오픈 소스 소프트웨어를 기반으로 한 방법이 더 널리 알려집니다. 생체 분석 소프트웨어를 사용하여 신중한 이미지 분석은 시각적 분석을 실체화하고 통계적 유의성을 부여 할 때 주어진 표현형의 정확성에 대한 신뢰도를 높이기 때문에 가치가 있습니다. 이전에는 SG가 무료 ImageJ 소프트웨어를 사용하여 분석되었으므로 개별 SG를 수동으로 식별해야했습니다 4 . 여기에 우리는 bac의 맥락에서 세포 SG 형성의 유도와 분석을위한 프로토콜을 제공한다.(http://icy.bioimageanalysis.org)를 사용하여 테리아 (terial) 감염을 예방할 수 있습니다. 바이오 이미지 분석 소프트웨어에는 SG 분석에 매우 적합한 스팟 디텍터 프로그램이 내장되어 있습니다. 지정된 관심 영역 (ROI)에서 자동화 된 탐지 프로세스를 미세 조정할 수 있습니다. 이는 개별 SG에 대한 수동 분석의 필요성을 극복하고 샘플링 편차를 제거합니다. 많은 환경 적 스트레스는 직경이 0.2 - 5 μm 인 상 고밀도 세포질, 비막 성 구조 인 SGs의 형성을 유도한다. 이 세포 반응은 효모, 식물 및 포유 동물에서 진화 적으로 보존되며 전 세계 단백질 번역이 금지 될 때 발생합니다. SG 로의 stalled translation initiation complex의 응집을 포함하며, SG는 translationally-inactive mRNA의 보유 장소로 간주되어 세포 mRNA의 일부를 선택적으로 번역 할 수있다.스트레스를 제거하면 SG가 분해되고 단백질 합성의 전체 속도가 재개됩니다. SG는 정확한 신랄한 구성이 적용되는 응력에 따라 달라질 수 있지만 번역 신생 개시 인자, RNA 대사에 관여하는 단백질, RNA 결합 단백질, 그리고 스 캐 폴딩 단백질 및 숙주 세포 신호 전달에 관련된 인자로 구성된다. SG 형성을 유도하는 환경 요인으로는 아미노산 결핍, 자외선 조사, 열 쇼크, 삼투압 쇼크, 소포체 세균 스트레스, 저산소증 및 바이러스 감염 2 , 7 , 8이있다 . 박테리아, 곰팡이 또는 원충 병원체와 같은 다른 병원체가이 세포 성 스트레스 반응에 어떻게 영향을 미치는지에 대해서는 아직 거의 알려지지 않았지만 바이러스가 유도하는 방법과 SG 형성을 파괴하는 방법에 대한 많은 진전이있었습니다. 시게lla flexneri는 사람의 그람 음성 음성 매개 성 세포질 병원체이며 심한 설사 또는 세균성 경화증의 원인균이다. 이질균증은 주요 공중 보건 부담이며 5 세, 9 세 이하의 어린이에서 매년 2 만 8 천명의 사망자를 발생시킵니다. S. flexneri 는 결장 상피를 감염시키고 숙주의 세포 골격 구성 요소 11 , 12 를 하이재킹하여 세포 대 세포를 퍼집니다. 상피의 감염은 세포질 내에서의 플레 서 네리 ( flexneri) 의 복제를지지하지만, 감염된 대식 세포는 열광 증 (pyroptosis)이라 불리는 염증 세포 사멸 과정을 통해 사망한다. 감염은 호중구와 열, 산화 스트레스 및 조직 파괴를 동반하는 심한 염증의 대규모 모집으로 이어집니다. 따라서, 감염된 세포는 골지사 파괴, 유전 독성 스트레스 및 세포 골격 재 배열과 같은 감염에 의해 유도 된 내부 스트레스를받는 반면감염된 세포는 또한 염증 과정으로 인해 환경 스트레스를받습니다. 다수의 SG 마커를 사용하여 환경 스트레스에 반응하는 세포의 능력에 대한 S. flexneri 감염의 특성화는 감염이 SG 조성 3의 질적 및 양적 차이를 유도한다는 것을 입증했다. 그러나 다른 박테리아 병원균에 대해서는 알려진 바가 거의 없습니다. 여기에서 우리는 cytosolic 병원체 S. flexneri , 다른 환경 스트레스와 세포의 스트레스, SG 구성 요소의 표시 및 감염의 맥락에서 SG 형성과 구성의 질적 및 정량 분석과 숙주 세포의 감염에 대한 방법론을 설명합니다 및 비 감염 세포. 이 방법은 다른 세균 병원균에 널리 적용 할 수 있습니다. 또한, SG 형성의 이미지 분석은 바이러스 또는 다른 병원균에 의한 감염에 사용될 수있다. SG를 분석하는 데 사용할 수 있습니다.감염시의 형성 또는 외인성 응력에 대한 SG 형성에 대한 감염의 효과.

<table><tbody><tr><td>&lt;strong&gt;Primary Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>eIF3b (N20), origin goat&amp;nbsp;</td><td>Santa Cruz</td><td>sc-16377</td><td>Robust and widely used SG marker. Cytosolic staining allows cell delineation. Dilution 1:300</td></tr><tr><td>eIF3b (A20), origin goat&amp;nbsp;</td><td>Santa Cruz</td><td>sc-16378</td><td>Same target as eIF3b (N20) and in our hands was identical to eIF3b (N20). Dilution 1:300</td></tr><tr><td>eIF3A (D51F4), origin rabbit (MC: monoclonal)</td><td>Cell Signaling</td><td>3411</td><td>Part of multiprotein eIF3 complex with eIF3b . Dilution 1:800</td></tr><tr><td>eIF4AI, origin goat&amp;nbsp;</td><td>Santa Cruz</td><td>sc-14211</td><td>Recommended by (Ref # 13). Dilution 1:200</td></tr><tr><td>eIF4B, origin rabbit</td><td>Abcam</td><td>ab186856</td><td>Good stress granule marker in our hands. Dilution 1:300</td></tr><tr><td>eIF4B, origin rabbit</td><td>Cell Signaling</td><td>3592</td><td>Recommended by Ref # 13. Dilution 1:100</td></tr><tr><td>eIF4G, origin rabbit</td><td>Santa Cruz</td><td>sc-11373</td><td>Widely used SG marker. (Ref # 13): may not work well in mouse cell lines. Dilution 1:300</td></tr><tr><td>G3BP1, origin rabbit (MC: monoclonal)</td><td>BD Biosciences</td><td>611126</td><td>Widely used SG marker. Dilution 1:300</td></tr><tr><td>Tia-1, origin goat&amp;nbsp;</td><td>Santa Cruz</td><td>sc-1751</td><td>Widely used SG marker. Can also be found in P bodies when SG are present (Ref # 13). Dilution 1:300</td></tr><tr><td>&lt;strong&gt;Alexa-conjugated Secondary Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>A488 anti-goat , origin donkey</td><td>Thermo Fisher</td><td>A-11055</td><td>Cross absorbed. Dilution 1:500</td></tr><tr><td>A568 anti-goat, origin donkey</td><td>Thermo Fisher</td><td>A-11057</td><td>Cross absorbed. Dilution 1:500</td></tr><tr><td>A488 anti-mouse, origin donkey</td><td>Thermo Fisher</td><td>A-21202</td><td>Dilution 1:500</td></tr><tr><td>A568 anti-mouse, origin donkey</td><td>Thermo Fisher</td><td>A10037</td><td>Dilution 1:500</td></tr><tr><td>A647 anti-mouse, origin donkey</td><td>Thermo Fisher</td><td>A31571</td><td>Dilution 1:500</td></tr><tr><td>A488 anti-rabbit, origin donkey</td><td>Thermo Fisher</td><td>A-21206</td><td>Dilution 1:500</td></tr><tr><td>A568 anti-rabbit, origin donkey</td><td>Thermo Fisher</td><td>A10042</td><td>Dilution 1:500</td></tr><tr><td>&lt;strong&gt;Other Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Shigella flexneri&lt;/em&gt;</td><td></td><td></td><td>Available from various laboratories by request</td></tr><tr><td>Tryptone Casein Soya (TCS) broth</td><td>BD Biosciences</td><td>211825</td><td>Standard growth medium for Shigella, application - bacterial growth</td></tr><tr><td>TCS agar</td><td>BD Biosciences</td><td>236950</td><td>Standard growth agar for Shigella, application - bacterial growth</td></tr><tr><td>Congo red</td><td>SERVA Electrophoresis GmbH</td><td>27215.01</td><td>Distrimination tool for Shigell that have lost the virulence plasmid, application - bacterial growth</td></tr><tr><td>Poly-L-Lysine (PLL)</td><td>Sigma-Aldrich</td><td>P1274</td><td>Useful to coating bacteria to increase infection, application - infection</td></tr><tr><td>Gentamicin</td><td>Sigma-Aldrich</td><td>G1397</td><td>Selective killing of extracellular but not cytosolic bacteria, application - infection</td></tr><tr><td>HEPES</td><td>Life Technologies</td><td>15630-056</td><td>PH buffer useful when cells are incubated at RT, application - cell culture</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Life Technologies</td><td>31885</td><td>Standard culture medium for HeLa cells, application - cell culture</td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>Biowest</td><td>S1810-100</td><td>5% supplementation used for HeLa cell culture medium, application - cell culture</td></tr><tr><td>Non-Essential Amino Acids (NEAA)</td><td>Life Technologies</td><td>11140</td><td>1:100 dilution used for HeLa cell culture medium, application - cell culture</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D2650</td><td>Reagent diluent, application - cell culture</td></tr><tr><td>Sodium arsenite</td><td>Sigma-Aldrich</td><td>S7400</td><td>Potent stress granule inducer (Note: highly toxic, special handling and disposal required), application - stress inducer</td></tr><tr><td>Clotrimazole</td><td>Sigma-Aldrich</td><td>C6019</td><td>Potent stress granule inducer (Note:health hazard, special handling and disposal required), application - stress inducer</td></tr><tr><td>Paraformaldehyde (PFA</td><td>Electron Microscopy Scences</td><td>15714</td><td>4% PFA is used for standard fixation of cells, application - fixation</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td>Used at 0.03% for permeabilizationof host cells before immunofluorescent staining, application - permeabilization</td></tr><tr><td>A647-phalloidin</td><td>Thermo Fisher</td><td>A22287</td><td>Dilution is at 1:40, best added during secondary antibody staining, application - staining</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td>Nucleid acid stain used to visualize both the host nucleus and bacteria, application - staining</td></tr><tr><td>Parafilm</td><td>Sigma-Aldrich</td><td>BR701501</td><td>Paraffin film useful for immunofluorescent staining of coverslips, application - staining</td></tr><tr><td>Prolong Gold</td><td>Thermo Fisher</td><td>36930</td><td>Robust mounting medium that works well for most fluorophores , application - mounting</td></tr><tr><td>Mowiol</td><td>Sigma-Aldrich</td><td>81381</td><td>Cheap and robust mounting medium that works well for most fluorophores, application - mounting</td></tr><tr><td>24-well cell culture plate</td><td>Sigma-Aldrich</td><td>CLS3527</td><td>Standard tissue culture plates, application - cell culture</td></tr><tr><td>12 mm&amp;nbsp; glass coverslips</td><td>NeuVitro</td><td>1001/12</td><td>Cell culture support for immunofluorescent applications, application - cell support</td></tr><tr><td>forceps</td><td>Sigma-Aldrich</td><td>81381</td><td>Cheap and robust mounting medium that works well for most fluorophores, application - mounting</td></tr><tr><td>&lt;strong&gt;Programs and Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Prism</td><td>GraphPad Software</td><td></td><td>Data analysisand graphing program with robust statistical test options, application - data analysis</td></tr><tr><td>Leica SP5</td><td>Leica Microsystems</td><td></td><td>Confocal microsope, application - image acquisition</td></tr><tr><td>Imaris</td><td>Bitplane</td><td></td><td>Professional image analysis program, application - data analysis</td></tr><tr><td>Excel</td><td>Microsoft</td><td></td><td>Data analysis and graphing program, application - data analysis</td></tr></tbody></table>

immunofluorescence analysis of stress granule formation after bacterial challenge of mammalian cells

세포 구성 요소의 형광 이미징은 숙주 - 병원체 상호 작용을 조사하는 효과적인 도구입니다. 병원균은 세포 소기관의 초 미세 구조, 세포 골격 네트워크 조직 및 스트레스 과립 (SG) 형성과 같은 세포 과정을 포함하여 감염된 세포의 다양한 특성에 영향을 줄 수 있습니다. 어떻게 병원균이 숙주 과정을 파괴하는지에 대한 특성 규명은 발병 기전의 중요하고 필수적인 부분이다. 다양한 표현형이 쉽게 볼 수 있지만, 병원균 공격에 의해 유발 된 세포 구조의 질적 및 양적 차이에 대한 정확한 분석은 실험 및 대조군 시료간에 통계적으로 중요한 차이를 정의하는 데 필수적입니다. SG 형성은 항 바이러스 반응을 유도하는 진화론 적으로 보전 된 스트레스 반응이며, 바이러스 감염을 이용하여 오랫동안 연구되어왔다. SG 형성은 또한 신호 계통에 영향을 미치고 다른 아직 알려지지 않은 conseq2 . 박테리아 병원균과 같은 바이러스 이외의 병원균에 대한 스트레스 반응의 특성화는 현재 연구의 새로운 영역 3 입니다. 현재 바이러스 성 시스템에서도 SG 형성에 대한 정량 분석과 정성 분석은 아직 일상적으로 사용되지 않고 있습니다. 여기서 우리는 다양한 외인성 응력에 대응하여 SGs의 형성에 영향을주는 세포 독성 세균 병원체에 감염된 세포 및 감염되지 않은 세포에서 SG 형성을 유도하고 특징 화하기위한 간단한 방법을 기술한다. SG 생성 및 구성 분석은 다양한 SG 마커와 오픈 소스 이미지 분석 도구 인 ICY의 스팟 디텍터 플러그인을 사용하여 이루어집니다.

이 원고에 설명 된 프로토콜을 설명하고 설명하기 위해 우리는 세포질 병원체 S. flexneri에 감염되었거나 감염되지 않은 HeLa 세포에서 clotrimazole 유도 된 SG의 이미지를 특징으로했습니다. 이 절차의 개요는 그림 1 에 나와 있으며 콩고 레드 플레이트에 박혀 있는 독성과 무해한 S. flexneri , 박테리아 준비, 감염, 환경 스트레스 추가, ?...

Watch this Scientific Journal Video about Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells at JoVE.com

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

우리는 세포가 박테리아와 여러 가지 스트레스에 도전을받은 후 포유류 세포에서 스트레스 과립 형성의 정성 및 정량 분석 ​​방법을 설명합니다. 이 프로토콜은 광범위한 호스트 - 박테리아 상호 작용에서 세포 스트레스 과립 반응을 조사하는 데 적용 할 수 있습니다.

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 ...

immunofluorescence-analysis-stress-granule-formation-after-bacterial

Research

JoVE Journal

Immunology and Infection

10.1K 조회수.  Institut Pasteur.  우리는 세포가 박테리아와 여러 가지 스트레스에 도전을받은 후 포유류 세포에서 스트레스 과립 형성의 정성 및 정량 분석 ​​방법을 설명합니다. 이 프로토콜은 광범위한 호스트 - 박테리아 상호 작용에서 세포 스트레스 과립 반응을 조사하는 데 적용 할 수 있습니다. 

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

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 ...

연구

면역학 및 감염병학

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

칩에 스트레스 유발 항생제 감수성 테스트

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

생체공학

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion (STED) Microscopy

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure that optimizes B-cell receptor (BCR) signaling and BCR-mediated antigen acquisition. Both the remodeling of the actin cytoskeleton and the reorientation of the microtubule network towards the antigen contact site are essential for immune synapse formation. Remodeling of the actin cytoskeleton into a dense peripheral ring of F-actin is accompanied by polarization of the microtubule-organizing center towards the immune synapse. Microtubule plus-end binding proteins, as well as cortical plus-end capture proteins mediate physical interactions between the actin and microtubule cytoskeletons, which allow them to be reorganized in a coordinated manner. Elucidating the mechanisms that control this cytoskeletal reorganization, as well as understanding how these cytoskeletal structures shape immune synapse formation and BCR signaling, can provide new insights into B cell activation. This has been aided by the development of super-resolution microscopy approaches that reveal new details of cytoskeletal network organization. We describe here a method for using stimulated emission depletion (STED) microscopy to simultaneously image actin structures, microtubules, and transfected GFP-tagged microtubule plus-end binding proteins in B cells. To model the early events in immune synapse formation, we allow B cells to spread on coverslips coated with anti-immunoglobulin (anti-Ig) antibodies, which initiate BCR signaling and cytoskeleton remodeling. We provide step-by-step protocols for expressing GFP fusion proteins in A20 B-lymphoma cells, for anti-Ig-induced cell spreading, and for subsequent cell fixation, Immunostaining, image acquisition, and image deconvolution steps. The high-resolution images obtained using these procedures allow one to simultaneously visualize actin structures, microtubules, and the microtubule plus-end binding proteins that may link these two cytoskeletal networks.

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy

We present a protocol for using STED microscopy to simultaneously image actin structures, microtubules, and microtubule plus-end binding proteins in B cells that have spread on coverslips coated with antibodies to the B-cell receptor, a model for the initial phase of immune synapse formation.

말라와 자극된 방출 소모 (호텔 위치) 현미경을 사용 하 여 B 세포 면역 냅에 Microtubule Cytoskeletons 시각화

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure ...

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, function, and fate in general. Although increasingly more adherent cell types&#39; responses to matrix stiffness have been characterized, how adherent cells&#39; susceptibility to bacterial infection depends on matrix stiffness is largely unknown, as is the effect of bacterial infection on the biomechanics of host cells. We hypothesize that the susceptibility of host endothelial cells to a bacterial infection depends on the stiffness of the matrix on which these cells reside, and that the infection of the host cells with bacteria will change their biomechanics. To test these two hypotheses, endothelial cells were used as model hosts and Listeria monocytogenes as a model pathogen. By developing a novel multi-well format assay, we show that the effect of matrix stiffness on infection of endothelial cells by L. monocytogenes can be quantitatively assessed through flow cytometry and immunostaining followed by microscopy. In addition, using traction force microscopy, the effect of L. monocytogenes infection on host endothelial cell biomechanics can be studied. The proposed method allows for the analysis of the effect of tissue-relevant mechanics on bacterial infection of adherent cells, which is a critical step towards understanding the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. This method is also applicable to a wide variety of other types of studies on cell biomechanics and response to substrate stiffness where it is important to be able to perform many replicates in parallel in each experiment.

We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria.

부착 세포의 세균성 감염에 기질 경직성의 영향을 공부 다 잘 형식 Polyacrylamide 기반 분석

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, ...

Substructure Analyzer: A User-Friendly Workflow for Rapid Exploration and Accurate Analysis of Cellular Bodies in Fluorescence Microscopy Images

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in live-cell imaging and high-throughput microscopy techniques. This led to a constant increase in the amount and complexity of the microscopy data for a single experiment. Because manual analysis of microscopy data is very time consuming, subjective, and prohibits quantitative analyses, automation of bioimage analysis is becoming almost unavoidable. We built an informatics workflow called Substructure Analyzer to fully automate signal analysis in bioimages from fluorescent microscopy. This workflow is developed on the user-friendly open-source platform Icy and is completed by functionalities from ImageJ. It includes the pre-processing of images to improve the signal to noise ratio, the individual segmentation of cells (detection of cell boundaries) and the detection/quantification of cell bodies enriched in specific cell compartments. The main advantage of this workflow is to propose complex bio-imaging functionalities to users without image analysis expertise through a user-friendly interface. Moreover, it is highly modular and adapted to several issues from the characterization of nuclear/cytoplasmic translocation to the comparative analysis of different cell bodies in different cellular sub-structures. The functionality of this workflow is illustrated through the study of the Cajal (coiled) Bodies under oxidative stress (OS) conditions. Data from fluorescence microscopy show that their integrity in human cells is impacted a few hours after the induction of OS. This effect is characterized by a decrease of coilin nucleation into characteristic Cajal Bodies, associated with a nucleoplasmic redistribution of coilin into an increased number of smaller foci. The central role of coilin in the exchange between CB components and the surrounding nucleoplasm suggests that OS induced redistribution of coilin could affect the composition and the functionality of Cajal Bodies.

We present a freely available workflow built for rapid exploration and accurate analysis of cellular bodies in specific cell compartments in fluorescence microscopy images. This user-friendly workflow is designed on the open-source software Icy and also uses ImageJ functionalities. The pipeline is affordable without knowledge in image analysis.

하위 구조 분석기: 형광 현미경 이미지에서 세포 체의 신속한 탐색 및 정확한 분석을 위한 사용자 친화적인 워크플로우

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in ...

Multi-scale Analysis of Bacterial Growth Under Stress Treatments

Analysis of the bacterial ability to grow and survive under stress conditions is essential for a wide range of microbiology studies. It is relevant to characterize the response of bacterial cells to stress-inducing treatments such as exposure to antibiotics or other antimicrobial compounds, irradiation, non-physiological pH, temperature, or salt concentration. Different stress treatments might disturb different cellular processes, including cell division, DNA replication, protein synthesis, membrane integrity, or cell cycle regulation. These effects are usually associated with specific phenotypes at the cellular scale. Therefore, understanding the extent and causality of stress-induced growth or viability deficiencies requires a careful analysis of several parameters, both at the single-cell and at the population levels. The experimental strategy presented here combines traditional optical density monitoring and plating assays with single-cell analysis techniques such as flow cytometry and real time microscopy imaging in live cells. This multiscale framework allows a time-resolved description of the impact of stress conditions on the fate of a bacterial population.

This protocol allows a time-resolved description of bacterial growth under stress conditions at the single-cell and the cell population levels.

스트레스 치료하에 세균 성장의 다단계 분석

Analysis of the bacterial ability to grow and survive under stress conditions is essential for a wide range of microbiology studies. It is relevant to ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

단일 효모 세포에서 MAPK 유도 표현의 시간적 정량화

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

생물학

Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly and fate is important to understand the cellular response to stress. The deficiency in key factors of SGs like G3BP (RasGAP SH3 domain Binding Protein) leads to developmental defects in mice&#160;and alterations of the Central Nervous System. To study the dynamics of SGs in cells from an organism, one can culture primary cells and follow the localization of a transfected tagged component of SGs. We describe time-lapse experiment to observe G3BP1-containing SGs in Mouse Embryonic Fibroblasts (MEFs). This technique can also be used to study G3BP-containing SGs in live neurons, which is crucial as it was recently shown that these SGs are formed at the onset of neurodegenerative diseases like Alzheimer&#39;s disease. This approach can be adapted to any other cellular body and granule protein component, and performed with transgenic animals, allowing the live study of granules dynamics for example in the absence of a specific factor of these granules.

Stress granules (SGs) are cytoplasmic RNA granules containing stalled ribonucleoprotein particles (RNPs), and important in cellular response to various stresses. Dynamics of SGs can be followed in live cells by visualizing the localization of a tagged component of SGs in transfected primary cells after stress.

라이브 일차 전지에 G3BP 스트레스 과립 역학의 시각화

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly ...

Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to stress conditions, are implicated in various aspects of cell metabolism and survival. SGs modulate cellular signaling pathways, post-transcriptional gene expression, and stress response programs. The formation of these mRNA-containing granules is directly connected to cellular translation. SG assembly is triggered by inhibited translation initiation, and SG disassembly is promoted by translation activation or by inhibited translation elongation. This relationship is further highlighted by SG composition. Core SG components are stalled translation pre-initiation complexes, mRNA, and selected RNA-binding Proteins (RBPs). The purpose of SG assembly is to conserve cellular energy by sequestering translationally stalled housekeeping mRNAs, allowing for the enhanced translation of stress-responsive proteins. In addition to the core constituents, such as stalled translation preinitiation complexes, SGs contain a plethora of other proteins and signaling molecules. Defects in SG formation can impair cellular adaptation to stress and can thus promote cell death. SGs and similar RNA-containing granules have been linked to a number of human diseases, including neurodegenerative disorders and cancer, leading to the recent interest in classifying and defining RNA granule subtypes. This protocol describes assays to characterize and quantify mammalian SGs.

Stress Granules (SGs) are nonmembranous cytoplasmic structures that form in cells exposed to a variety of stresses. SGs contain mRNAs, RNA-binding proteins, small ribosomal subunits, translation-related factors, and various cell signaling proteins. This protocol describes a workflow that uses several experimental approaches to detect, characterize, and quantify bona fide SGs.

포유류 스트레스 과립으로 세포질 초점을 분류하는 방법

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to ...

An Immunofluorescence Assay to Characterize and Quantify Mammalian Stress Granules

Source: Aulas, A. et al., <a href="https://app.jove.com/v/55656">Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates an assay for characterizing and quantifying mammalian stress granules, which consist of mRNAs, RNA-binding proteins (RBPs), and various other proteins.

포유류 스트레스 과립을 특성화하고 정량화하기 위한 면역형광 분석

1. Cell Preparation

	Add autoclaved coverslips to 12 wells of a 24-well plate, as indicated in&#160;Figure 1A; this can be done using a sterile Pasteur ...