본 연구는 보건 국립 연구소에서 그랜트와 알링턴 텍사스 대학 MM에 기금을 설치하여 지원했다.

1. 성장하는 식물과 박테리아 준비 <ol><li> V : 성장 매체의 V 혼합물 (Redi - 지구 플러그 앤 세들링 믹스, 일요일 Gro), 고급 vermiculite와 펄라이트이 절차를 시작하려면 1시 1분 1초 V에 Arabidopsis 씨를 뿌리다. </li><li> 성장 챔버 (22 ° C, 100 μmol / mm 2 / 초 매일 가벼운 65 12 시간의 ± 5 % 습도)와 필요에 따라 물속에 식물을 성장. 식물들은 이전 탈당 젊은 완전히 확장 나뭇잎이있을 때 4~6주에서 사용할 준비가 된 것입니다. </li><li> 실험을 시작하기 전에 이틀 세균 문화를 준비합니다. 수정된 LB 매체 (10 G / L tryptone, 5 G / L 효모 추출물, 5 G / L NaCl 산도 7.0 및 1.5 %의 한천) 및 28에서 24 시간 동안 품어 ° C.에 글리세롤 주식의 리크 모나스 syringae inoculum를 준비하고 박테리아가 하위 culturing 후 적은 악성되면서 항상 글리세롤 주식의 문화 접시를 시작하는 새로운 문화를 사용합니다. </li><li> P.의 10 ML 액체 문화를 시작 접시에 자란 박테리아를 사용하여 50 ML Erlenmeyer 플라스크에 syringae. 28 밤새 문화를 품어 ° C가 0.8 일 사이에 600 nm의에서 광학 밀도 또는 OD에 도달할 때까지 활발한 떨고와 함께. </li><li> 600에서 OD를 측정하고 10 분 동안 1,360 XG에서 원심 분리하여 세포를 수확. 최종 OD가 0.2이되도록 증류수에있는 세포를 Resuspend. 이 OD는 10 8 콜로니 형성 단위 (CFU) / ML에 해당합니다. 준비 박테리아와 나뭇잎을 준비하고 분석을 수행하기 위해 진행합니다. </li></ol> 2. 박테리아와 리프 스테 이닝과 부화 <ol><li> 단풍은 처음 20 μm의의 propidium 요오드화물의 또는 물에 PI 솔루션을 준비 얼룩하기​​ 위해서. 솔루션의 10 ML는 한 번에 세 작은 잎을 얼룩이 충분하다. </li><li> 세 젊은 완벽하게 확장 단풍 성장 챔버로부터 포셉 분리와 함께 식물을 검색할 수 있습니다. 5 분 PI 솔루션에있는 모든 나뭇잎을 담가. 그런 나뭇잎을 제거하고 증류수로 잠시 그​​들을 씻어. </li><li> 현미경 슬라이드에 아래로 직면하고있는 낮은 표면 다음 장소는 나뭇잎. 나뭇잎이 슬라이드 평면 낳는 있도록 중간 정맥을 제외한 네 조각으로 날카로운 면도날로 나뭇잎을 잘라. </li><li> 나뭇잎과 박테리아를 부화, 슬라이드에있는 잎 조각 아래의 세균 현탁액 300 μL를 추가합니다. 잎사귀의 낮은 표면 inoculum 접촉되어 있는지 확인합니다. </li><li> 식물을 성장하는 데 사용된 동일한 환경 조건에서 샘플을 품어. 현미경으로 나뭇잎을 관찰하기 위해 당시까지 직면 낮은 표면을 가진 새 슬라이드에 잎 조각을 전송할 수 있습니다. 슬라이드를 장착하면 커버 용지가 사용되지 않습니다. 같은 잎 샘플 부화하는 동안 여러 번 몇 군데있을 수 있으며 시간 과정은 연구 목적에 따라 설정할 수 있습니다. </li></ol> 3. 현미경, 측정 및 데이터 분석 <ol><li> 여기 공촛점 현미경 스캐닝 레이저 (LSM 510 메타, 칼 자이스 혈구 주)는 나뭇잎을 검사하는 데 사용됩니다. PI (453 nm의 여기, 방사 543 및 620 nm의)의 형광을 감지하기 위해 나뭇잎의 낮은 표면을 관찰. </li><li> 같은 잎 샘플을 사용하면 시간이 지남에 따라 stomata의 이미지를 캡처합니다. stomatal 조리개의 넓이를 측정하기위한 이미지를 저장합니다. </li><li> LSM 이미지 브라우저를 사용하여 각 시점에서 각 치료를 위해 최소 60 stomata의 stomatal 조리개 너비를 측정합니다. </li><li> stomatal 개구 폭에 대한 평균과 표준 오차를 계산. 결과의 통계적 의의 2 - 꼬리, 이점 현명한 학생의 T - 테스트를 사용하여 계산하실 수 있습니다. </li></ol> 4. 박테리아 배양에 Stomatal 답변 대표 이미지 <ol><li> 여기 밝은 필드 뷰 (그림 1)에 공촛점 현미경 스캐닝 레이저 아래 Arabidopsis 잎 표면의 전형적인 미세한 이미지 (20x 목표를 사용)입니다. </li><li> Propidium 요오드화물 얼룩의 데이터 수집 (그림 2)에 대한 손상이 세포의 식별을 허용 이외에 경비 세포의 가시성을 증가 가능한 세포의 세포 벽. stomatal 구멍 구멍의 범위는 완전히 폐쇄 stomata에서 활짝 열려 stomata하려면 다음 micrographs에서 볼 수 있습니다. </li><li> 완전히 폐쇄 stomata은 경비 세포의 모양 (그림 3)에 의해 식별됩니다. 개구 폭 0 μm의 것으로 간주됩니다. </li><li> 오픈 stomata (그림 4)와 같이 조리개 너비가 stomatal 기공의 넓은 지역에 걸쳐 그려진 직선을 사용하여 측정됩니다. </li><li> 이들은이 stomatal 분석의 전형적인 실험 결과입니다. Arabidopis 단풍은 모나스 syringae의 세 가지 다른 변종과 함께 어둠 속에서 incubated되었습니다. 만이 세균은 PST DC3000은 박테리아 스트레인마다 무작위로 선택된 60 stomata (그림 5)의 개구 폭에 의해 측정 어두운 폐쇄 stomata를 열 수있었습니다. </li></oL&gt; <img src="/files/ftp_upload/2185/2185fig1.jpg" alt="그림 1" /> 그림 1. Arabidopsis 잎 표면의 현미경. 20x 목표를 사용 공촛점 현미경 (LSCM)를 스캐닝 레이저에서 잎 표면의보기 중 하나는 분야. propidium 요오드화물 얼룩 (그림 2)에 의해 주었 형광등 볼 수 비교할 때 stomatal 조리개과 같이 분명되지 않습니다. <img src="/files/ftp_upload/2185/2185fig2.jpg" alt="그림 2" /> 그림 2. propidium 요오드화물 - 스테인드 Arabidopsis 리프의 표면의 현미경. 20x 목표를 사용 공촛점 현미경 (LSCM)를 스캐닝 레이저에서 잎 표면의보기 중 하나는 분야. stomatal 기공 개방의 범위를합니다. 노란색 화살표는 완전히 stomata를 닫고 지적하고 녹색 화살표가 활짝 열려 stomata 지적하고 있습니다. <img src="/files/ftp_upload/2185/2185fig3.jpg" alt="그림 3" /> 그림 3. 완전의 모양과 경비 세포 사이의 개방으로 식별 가까이 stomata가. 개구 폭은 0 μm의 것으로 간주됩니다. <img src="/files/ftp_upload/2185/2185fig4.jpg" alt="그림 4" /> 그림 4. μm의에서 조리개 너비를 보여주는 오픈 stomata가. 측정은 stomatal 기공의 넓은 지역에 걸쳐 그린 직선에 따라 LSCM 브라우저를 사용하여 촬영되었다. <img src="/files/ftp_upload/2185/2185fig5.jpg" alt="그림 5" /> 그림 5. .. 세 모나스 syringae PV 토마토의 변종과 함께 incubated Arabidopsis 나뭇잎의 Stomatal 조리개 : DC3118, DB29, 그리고 DC3000 공장은 실험 시간 동안 어둠 속에 보관했다. 결과는 의미로 표시됩니다 (N = 50-70) ± 표준 오차. 통계 중요성은 두 꼬리 학생의 T - 테스트를 발견했습니다. 실험과 유사한 결과를 세 번 반복되었다.

<ol>
	<li>Brooks, D. M., Hernandez-Guzman, G., Kloek, A. P., Alarcon-Chaidez, F., Sreedharan, A., Rangaswamy, V., Penaloza-Vazquez, A., Bender, C. L., Kunkel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+well-defined+series+of+coronatine+biosynthetic+mutants+of+Pseudomonas+syringae+pv.+tomato+DC3000.">Identification and characterization of a well-defined series of coronatine biosynthetic mutants of Pseudomonas syringae pv. tomato DC3000.</a> Mol Plant-Microbe Interact. 17, 162-174 (2004).</li><li>Katagiri, F., Thilmony, R., He, S. Y., Somerville, C. R., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+thaliana-Pseudomonas+syringae+interaction.">The Arabidopsis thaliana-Pseudomonas syringae interaction.</a> The Arabidopsis Book. , (2002).</li><li>Ma, S. W., Morris, V. L., Cuppels, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+DNA+region+required+for+production+of+the+phytotoxin+coronatine+by+Pseudomonas+syringae+pv.+tomato.">Characterization of a DNA region required for production of the phytotoxin coronatine by Pseudomonas syringae pv. tomato.</a> Mol Plant-Microbe Interact. 4, 69-74 (1991).</li><li>Melotto, M., Underwood, W., Koczan, J., Nomura, K., He, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+stomata+function+in+innate+immunity+against+bacterial+invasion.">Plant stomata function in innate immunity against bacterial invasion.</a> Cell. 126, 969-980 (2006).</li></ol>

관심 없음 충돌 선언하지 않습니다.

우리는 다른 치료에 반응 stomatal 쉽게 평가 있도록 그대로 잎 조직에서 stomatal 조리개를 측정하는 직선 전달 절차를 제시합니다. 우리가 모델 시스템 Arabidopsis / 모나스 syringae를 사용하여 결과를 발표했지만, 손상 잎 stomatal 분석은 잠재적으로 어떤 식물 세균의 조합을 수행할 수 있습니다. 프로토콜은 쉽게 다른 식물과 세균성 병원균의 성장 요구에 맞게 수정할 수 있습니다. 전체 원칙과 절차는 동일하게 유지. 또한,이 방법은 미생물을 살아 경호 세포뿐만 아니라 출력의 기능을 연구하고자하는 연구자에게 도움이 될뿐 아니라 나뭇잎의 자연 환경을 유지 조건에서 다른 자극과 화학 약품에 있습니다. 전체 잎의 두께와 고르지 지형으로 인해 이미지에 어려울 수 있습니다. 이 문제는 그것이 평면 낳는 있도록 슬라이드에 나뭇잎의 중간 정맥을 제거하고 공촛점 현미경을 사용하여 완화 할 수 있습니다. 그러나, 형광 현미경의 다른 종류는 잎 표면의 고해상도 이미지를 얻기 위해 사용할 수 있습니다.

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Propidium iodide</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>P4864</td>
<td>&nbsp;</td>
<tr>
<td>Tryptone</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP1421-1</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Extract</td>
<td>&nbsp;</td>
<td>Difco</td>
<td>0127-01-7</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>7647-14-5</td>
<td>&nbsp;</td>
<tr>
<td>Agar</td>
<td>&nbsp;</td>
<td>Bio Express</td>
<td>J637-2500G</td>
<td>&nbsp;</td>
<tr>
<td>Plant growth chamber</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Shaker incubator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Laser scanning confocal microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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assessing stomatal response to live bacterial cells using whole leaf imaging

Stomata은 공장 내부와 환경 사이의 가스 교환에 책임이있는 식물 표피 자연 채용하고 있습니다. 그들은 빛의 강도, 이산화탄소 농도, 그리고 상대 습도 (RH)를 포함한 외부 요인에 대응 stomatal 기공을 닫으 수있는 경비 세포, 한 쌍의에 의해 형성된다. stomatal 기공은 나뭇잎으로 병원체 진입, 질병 개발을위한 중요한 단계의 주요 경로입니다. 최근 연구는 기공의 폐쇄가 Arabidopsis 식물의 세균성 질병 개발을 최소화에 효과적이라는 사실을 발표했습니다; 공장 타고난 면역의 중요한 부분을. 이전, 우리는 박테리아를 (. Melotto 외 2006) 생활 stomatal 반응을 평가하기 위해 표피 껍질을 사용한 적이 있지만 식물 표피 껍질을 세균 세포 모두에 유리한 환경 조건을 유지 도전했습니다. 잎 표피는 경비 세포 electrophysiological 실험 MES 버퍼 (10 MM KCl, 25 MM MES - 코, 산도 6.15)와 살아 있고 건강하게 만들어 줬어 수 있습니다. 그러나,이 버퍼가 박테리아 중지를위한 적합하지 않습니다. 한편, 세균 세포는 시간의 오랜 기간 동안 표피 껍질을 유지하기 위해 적절한되지 않습니다 물이 살아 보관하실 수 있습니다. 물 위에 표피 필 수레, 4 시간 이내에 건조 공기에 노출되어있는 껍질에있는 세포가 실험을 실시하는 시간을 제한하는 경우. 경비 세포에 특정 자극의 효과를 평가하기위한 이상적인 방법은 stomatal 생리학과 가능한 많은 식물의 자연 환경에 최소한의 간섭을 제시해야합니다. 우리는 그러므로, 잎 입힐 수있는 총알과 조작​​이 크게 쉽게 재현할하고 안정적​​인 stomatal 분석을 제공하는 목표로 최소화되는 박테리아를 살 stomatal 응답을 평가하는 새로운 방법을 개발했습니다. 프로토콜은 propidium 요오드화물의 (PI), 세균의 정지와 리프 얼룩의 부화하고, 공촛점 현미경 스캐닝 레이저에 따라 나뭇잎의 관찰과 함께 그대로 잎의 얼룩을 기반으로합니다. 마지막으로,이 방법은 밀접하게 식물이 병원균에 의해 공격하는 아래의 자연 조건을 모방 조건을 사용하여 오랜 기간에 걸쳐 동일한 라이브 잎 샘플의 관찰을 허용합니다.

Watch this Scientific Journal Video about Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging at JoVE.com

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

우리는 사는 박테리아에 stomatal 응답을 액세스하는 간단하고 재현성 프로토콜을 개발했습니다. 이 방법은 표피 껍질의 사용에 비해 잎사귀의 입힐 수있는 총알과 조작​​은 이전에 보고된 최소화합니다.

전체 리프 이미징을 사용하여 박테리아 세포를 라이브 Stomatal 응답을 평가

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

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JoVE Journal

Immunology and Infection

18.2K 조회수.  University of Texas at Arlington. 우리는 사는 박테리아에 stomatal 응답을 액세스하는 간단하고 재현성 프로토콜을 개발했습니다. 이 방법은 표피 껍질의 사용에 비해 잎사귀의 입힐 수있는 총알과 조작​​은 이전에 보고된 최소화합니다.

비디오: Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

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

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

라이브 세포 형광 현미경을 사용하여 박테리아 독소 유도 응답의 시각화

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

발광 생물 세균 이미징<em&gt; 생체 내</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

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

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

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

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

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

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

리스테리아 단세포 유전자를 사용하여 감염된 마우스에 있는 문화와 심혼 식민지에 있는 심장 세포의 세균성 침략 평가

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

을 사용해서 식물 방어 응답의 미세 특성에 대한 세균성 잎 침투 분석<em&gt; 애기 장대-슈도모나스 시린 개 (Pseudomonas syringae)</em&gt; Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

의 생리적 반응을 측정<em&gt; 초파리</em&gt; 라이브 칼슘 이미징을 사용하여 지질 페로몬에 감각 뉴런

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

에 대한 응답으로 인간의 기본 호중구와 단핵구에 의해 항진균 활성의 라이브 영상<em&gt;를했다. 투스</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

과일 파리, 초파리 melanogaster에 비보 먹어서 분석 결과 사용 하 여의 세포 면역 반응 평가

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

정확한 세포 표현과 정확한 단백질 국소화를 위한 세균 세포의 3차원 이미징

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be ...