Bu araştırma, Ulusal Sağlık Enstitüsü ve The University of Texas at Arlington MM fonları tarafından hibe destek verdi.

1. Büyüyen Bitkiler ve hazırlanması Bakteriler <ol><li> V: v büyüyen orta karışımı (Redi-toprak fiş ve fide karışımı Sun, Gro), ince vermikülit, perlit, bu yordamı başlamak için 01:01:01 v Arabidopsis tohumları eker . </li><li> Bir büyüme odası (22 ° C, 12 saat 100 mmol / mm 2 / sn günlük ışık ve 65 ±% 5 nem) ve gerektiği kadar su bitkileri yetiştirin. Bitkiler 4-6 hafta içinde tamamen genişletilmiş genç yaprakları önce civatalama zaman kullanıma hazır. </li><li> Deney başlamadan önce iki gün bakteri kültürü hazırlar. LB, orta (10 g / L Tripton, 5 g / L maya özü, 5 g / L NaCl, pH 7.0 ve% 1.5 agar) değiştirilmiş ve 28 az 24 saat süreyle inkübe ° C gliserol stok Streak Pseudomonas syringae Inokulum hazırlamak ve bakteriler alt-kültür sonra daha az öldürücü olur gliserol stokları her zaman olduğu gibi kültür plakaları başlatmak için taze kültür kullanın. </li><li> Plaka üzerinde büyüdü bakteri P. 10 ml sıvı kültürü başlatmak için kullanın syringae 50 ml Erlenmeyer balonuna. Gecede kültür 28 ° C&#39;de 0.8 ile 1 arasında 600 nm dalga boyunda optik yoğunluk veya OD ulaşıncaya kadar kuvvetli sallayarak. </li><li> 600 OD ölçün ve 10 dakika 1360 xg&#39;de santrifüj hücrelerin hasat. Distile su ile son OD 0.2, böylece hücreleri tekrar Bu OD 10 8 koloni oluşturan birim (CFU) / mL karşılık gelir. Hazır bakteri, yaprakları hazırlamak ve analiz gerçekleştirmek için devam edin. </li></ol> 2. Bakteriler Yaprak Boyama ve Kuluçka <ol><li> Yaprakları ilk 20 mcM propidium iyodür veya suya PI çözüm hazırlamak leke için. 10 ml çözelti, bir seferde 3 küçük yaprak leke için yeterli. </li><li> 3 genç, tam genişletilmiş yaprak büyüme odasından ve forseps detach ile bitkiler al. 5 dakika PI çözüm tüm yaprakları bırakın. Sonra yaprakları kaldırmak ve onları saf su ile kısa bir süre yıkayın. </li><li> Sonraki yerde bir mikroskop lamı üzerine aşağı bakacak şekilde alt yüzeye sahip bir yaprak. Slayt yaprak düz bırakır, böylece orta damar içine keskin bir jilet hariç dört adet yaprak kesin. </li><li> Yaprakları ile bakteri inkübasyon için, slayt yaprak parçaları altında bakteriyel süspansiyon 300 mcL ekleyin. Yaprağın alt yüzeyi inokulum ile temas halinde olduğundan emin olun. </li><li> Bitkilerin büyümesi için kullanılan aynı çevre koşullarında örnekleri inkübe edin. Mikroskop altında yaprakları gözlemlemek zamanda yaprak parçaları, alt yüzeyi yukarı bakacak şekilde yeni bir slayt aktarın. Slaytlar montaj kapağı kayma olmadığını unutmayın. Aynı yaprak örneği inkübasyon sırasında birden çok kez görüntülenmiş olabilir ve bir süre ders, araştırma hedeflerine göre ayarlanmış olabilir. </li></ol> 3. Mikroskopi, Ölçme ve Veri Analizi <ol><li> Burada konfokal mikroskop bir lazer tarama (EKK 510 Meta, Carl Zeiss Inc.) Yaprakları incelemek için kullanılır. PI (eksitasyon 453 nm, emisyon 543 ve 620 nm) floresan algılamak için yaprakların alt yüzeyi dikkat edin. </li><li> Aynı yaprak örnekleri kullanarak zamanla stoma görüntüleri yakalayabilir. Stoma diyafram genişliğini ölçmek için kaydedin. </li><li> LSM görüntü tarayıcısı kullanarak her zaman bir noktada her bir tedavi için en az 60 stoma stoma diyafram genişliği ölçün. </li><li> Stoma diyafram genişliği için ortalama ve standart hata hesaplayın. Sonuçlarının istatistiksel anlamlılık 2 kuyruklu bilge Öğrenci, eşleştirilmiş t-testi kullanılarak hesaplandı. </li></ol> 4. Bakteriler Kuluçka Stoma Tepki Temsilcisi Görüntüler <ol><li> İşte, parlak bir alan görünümü (Şekil 1) konfokal mikroskop lazer tarama altında bir Arabidopsis yaprak yüzeyi tipik bir mikroskobik görüntü (20x amacı kullanarak) . </li><li> Propidium iyodür lekeleri veri toplama (Şekil 2) hasarsız hücrelerinin tanımlanması için izin vermeye ek olarak bekçi hücreleri görünürlüğünü artırarak canlı hücreleri hücre duvarı. Stoma gözenek açıklıkları bir dizi, tamamen kapalı stoma geniş açık stoma, bu mikrograflar görülebilir. </li><li> Tamamı kapalı stoma bekçi hücrelerin şekil (Şekil 3) tarafından tespit edilir. Diyafram genişliği 0 mm olarak kabul edilir. </li><li> Açık stoma (Şekil 4) için gösterilen diyafram genişliği stoma gözenek geniş alan boyunca çizilen düz bir çizgi ile ölçülür. </li><li> Bunlar bu stoma testin tipik deneysel sonuçlar Arabidopis yaprakları Pseudomonas syringae üç farklı suşları ile karanlıkta inkübe edildi. Sadece bakteri Pst DC3000 koyu-kapalı stoma, bakteri suşunun başına rastgele seçilen 60 stoma (Şekil 5) diyafram genişliği ile ölçülen açmak mümkün. </li></ol&gt; <img src="/files/ftp_upload/2185/2185fig1.jpg" alt="Şekil 1" /> Şekil 1. Arabidopsis bir yaprak yüzeyi Mikrografik konfokal mikroskobu (LSCM) 20x objektif kullanarak lazer tarama altında yaprak yüzeyi görüş alanı. Propidium iyodür boyama (Şekil 2) yardımıyla floresan görünümüne göre stoma diyafram gibi belirgin olmadığını unutmayın. <img src="/files/ftp_upload/2185/2185fig2.jpg" alt="Şekil 2" /> Şekil 2. Propidium iyodür lekeli Arabidopsis yaprak yüzeyi Mikrografik konfokal mikroskobu (LSCM) 20x objektif kullanarak lazer tarama altında yaprak yüzeyi görüş alanı. Stoma gözenek açılması bir dizi dikkat edin. Sarı oklar stoma tamamen kapatmak için işaret ve yeşil okları geniş açık stoma işaret edilir. <img src="/files/ftp_upload/2185/2185fig3.jpg" alt="Şekil 3" /> Şekil 3. Tamamen şekil ve bekçi hücreleri arasındaki açıklık tespit yakın stoma diyafram genişliği 0 mm olarak kabul edilir. <img src="/files/ftp_upload/2185/2185fig4.jpg" alt="Şekil 4" /> Şekil 4. Mikron diyafram genişliği gösteren Açık stoma. Ölçümler stoma gözenek geniş alan boyunca çizilen düz bir çizgi dayalı LSCM tarayıcı kullanarak alındı. <img src="/files/ftp_upload/2185/2185fig5.jpg" alt="Şekil 5" /> Şekil 5. Arabidopsis yaprakları üç Pseudomonas syringae pv domates suşları ile inkübe Stoma diyafram: DC3118, DB29 ve DC3000 Tesisleri deneme süresi boyunca karanlıkta tutulmuştur. Sonuçlar ortalama olarak verilmiştir (n = 50-70) ± standart hata. Iki kuyruklu Student t-testi ile istatistiksel anlamlılık tespit edildi. Deney benzer sonuçlar veren üç kez tekrarlandı.

<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>

Biz, farklı tedavilere stoma yanıt kolay bir değerlendirme için izin sağlam yaprak dokusunda stoma diyafram ölçmek için bir yalındır prosedürü sundu. Model sistem Arabidopsis / Pseudomonas syringae kullanarak sonuçlarını sundu olmasına rağmen, bozulmamış yaprak stoma tahlil potansiyel olarak herhangi bir bitki-bakteri kombinasyonu ile yapılabilir . Protokolü, diğer bitkiler ve bakteriyel patojenler büyüme gereksinimlerini kolayca sığacak şekilde modifiye edilebilir. Genel ilke ve prosedür aynı kalır. Buna ek olarak, bu yöntem mikroplar yaşamak için bekçi hücreleri sadece fonksiyonel çıktı okumak isteyen araştırmacılar için yararlı olabilir, ama aynı zamanda, yaprak doğal ortamını korumak koşullar altında diğer uyaranların ve kimyasal ajanlar olabilir. Tüm yaprak kalınlığı ve engebeli topografyası nedeniyle görüntü için zor olabilir. Bu sorun, yaprak orta damar düz durduğundan slayt kaldırma ve konfokal mikroskopi kullanılarak kontrol altına alınabilir. Ancak, diğer tip floresan mikroskop yaprak yüzeyinin yüksek çözünürlüklü görüntüleri elde etmek için kullanılabilir.

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

Stoma bitki iç ve çevre arasındaki gaz değişimi için sorumlu olan bitki epidermisin doğal açıklıklar. Bu ışık yoğunluğu, karbon dioksit konsantrasyonu, ve bağıl nem (RH) de dahil olmak üzere bir dizi dış etkenlere yanıt olarak stoma gözenek kapatmak için bekçi hücreleri, bir çifti tarafından oluşturulur. Stoma gözenek da ana güzergah yaprakların içine patojen giriş, hastalık gelişimi için önemli bir adım. Son çalışmalar, gözenek kapatılması Arabidopsis bitkilerin bakteriyel hastalık gelişimi en aza indirilmesinde etkili olduğunu açıkladı; bitki doğal bağışıklık ayrılmaz bir parçasıdır . Önceden, canlı bakteriler (Melotto et al 2006), stoma tepki değerlendirmek için epidermal peeling; ancak bitki epidermal peeling ve bakteri hücreleri için olumlu çevre koşulları muhafaza zorlu olmuştur . Yaprak epidermisin bekçi hücreleri elektrofizyolojik deneyler için MES tamponu (10 mM KCl, 25 mM MES-KOH, pH 6.15) ile canlı ve sağlıklı olarak muhafaza edilebilir. Ancak, bu tampon bakteri süspansiyonu elde etmek için uygun değildir. Öte yandan, bakteri hücrelerinin epidermal peeling kez uzun süre korumak için uygun değildir su canlı tutulabilir. 4 saat içinde hava kuru maruz kalan kabuğu, su üzerinde epidermal kabuğu yüzer, hücreleri deney yapmak için zamanlama sınırlayıcı. Bekçi hücreleri belirli bir uyaranın etkisini değerlendirmek için ideal bir yöntem stoma fizyolojisi ve tesisi mümkün olduğunca doğal çevreye minimal girişim sunmalıdır. Bu nedenle, yaprak yaralama ve manipülasyon kolayca tekrarlanabilir ve güvenilir bir stoma tahlil sağlamayı amaçlayan önemli ölçüde minimize edildiği bakterilerin yaşaması için stoma yanıtı değerlendirmek için yeni bir yöntem geliştirdi. Protokol propidium iyodür (PI), bakteri süspansiyonu ile yaprak boyama inkübasyon, lazer tarama konfokal mikroskop altında yaprakları gözlem ve sağlam yaprak boyama dayanmaktadır. Son olarak, bu yöntem bitkiler patojenler tarafından saldırıya altında doğal koşulları yakından taklit koşulları kullanarak uzun süre boyunca aynı canlı yaprak örnek gözlem için izin verir.

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

Biz canlı bakteri stoma yanıt erişmek için basit ve tekrarlanabilir bir protokol geliştirmiştir. Bu yöntem daha önce bildirdiği epidermal peeling kullanımı ile karşılaştırıldığında yaprak yaralama ve manipülasyona en aza indirir.

Tüm Yaprak Görüntüleme kullanarak Bakteriyel Hücreler Live Stoma Tepki Değerlendirilmesi

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

assessing-stomatal-response-to-live-bacterial-cells-using-whole-leaf

Research

JoVE Journal

Immunology and Infection

18.2K Görüntüleme.  University of Texas at Arlington. Biz canlı bakteri stoma yanıt erişmek için basit ve tekrarlanabilir bir protokol geliştirmiştir. Bu yöntem daha önce bildirdiği epidermal peeling kullanımı ile karşılaştırıldığında yaprak yaralama ve manipülasyona en aza indirir.

Video: 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.

Farelerde Görüntü Bakteriyel Enfeksiyonlar Lusiferaz Kullanımı

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

İmmünoloji ve Enfeksiyon

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.

Canlı Hücre Floresan Mikroskopi kullanma Bakteriyel Toksin İsteyerek Yanıtların Görselleştirme

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.

Bioluminescent Bakteriyel Görüntüleme<em&gt; In Vivo</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.

Fotoaktive Tek molekül Takip Kullanma Canlı Bakteriyel Hücrelerde Protein-DNA Etkileşimleri görselleştirme

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.

Listeria monocytogenes Kullanarak Enfekte Farelerde Kültür ve Kalp Kolonizasyonunda Kardiyak Hücrelerin Bakteriyel İstilasının Değerlendirilmesi

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.

Kullanarak Bitki Savunma Yanıtları ince Karakterizasyonu için Bakteriyel Yaprak Sızma Deneyi<em&gt; Arabidopsis thaliana-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.

Fizyolojik Yanıtları Ölçüm<em&gt; Drosophila</em&gt; Canlı Kalsiyum Görüntüleme kullanma Lipit Feromonlar Sensory Nöronlar

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.

Cevabı İnsan İlköğretim nötrofiller ve monositler tarafından Antifungal Faaliyet Canlı Görüntüleme<em&gt; A. fumigatus</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.

Bir In Vivo fagositoz tahlil kullanarak Drosophila melanogaster, meyve sineği hücresel bağışıklık yanıtı değerlendirirken

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.

Doğru Hücresel Temsiller ve Hassas Protein Lokalizasyonu için Bakteri Hücrelerinin Üç Boyutlu Görüntülemesi

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 ...