המחברים מבקשים להודות תמיכה רלוונטית לכתב היד הזה מהוועדה השביעית מסגרת התכנית האירופית (PIOF-GA-2009-255466) והמועצה למחקר הבריאות האירית (HRA_POR/2010/138). Lux מתויג-E. coli היה מתנה מסוג ד&quot;ר קורמאק Gahan, קורק מכללת האוניברסיטה.

1. אינדוקצית גידול <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לזירוז גידול שגרתי, המינון המינימאלי של תאים הסרטניים מושעים ב200 μl של התרבות בינונית סרום ללא הוזרק תת עורית (SC) לתוך האגף של זיהום החופשי בשבוע 6-8 עכברי BALB / C או athymic נשיים ישנים MF1-nu/nu n = 6 (הרלן, אוקספורדשייר, בריטניה) (1 x 10 6 תאים 4T1) באמצעות מחט מזרק 21-מד. הכדאיות של תאים המשמשים לחיסון הייתה גבוה מ 95%, כפי שנקבע על ידי ספירה חזותית באמצעות haemocytometer והדרת צבע הכחול Trypan (Gibco). </li><li style=";text-align:right;direction:rtl"> בעקבות הקמת גידול, גידולים הורשו לגדול ולהתפתח והיו במעקב פעמים בשבוע. נפח גידול היה מחושב לפי נוסחת V = (ab 2) Π / 6, שבו הוא הארוך ביותר בקוטר של גידול ואילו b הוא ניצב הקוטר הארוך ביותר לקוטר. </li></ol> 2. הכנת קטריאלי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> זן החיידקים המשמש בprotoco זהאני היה א coli K-12 MG1655, שאינו החלבון הרעלן-להביע מתח, מחסה luxABCDE קידוד פלסמיד המאפשר לחיידקים להיות מזוהים על ידי BLI. א coli MG1655 המכיל luxABCDE המשולב היה גדל אירובי על 37 מעלות צלזיוס במדיום LB (סיגמה אולדריץ, אירלנד) בתוספת 300 מיקרוגרם / המ&quot;ל אריתרומיצין (EM). הנגזרת של bioluminescent MG1655 נוצרה באמצעות p16S לוקס פלסמיד המכיל את עזרת P המכוננת luxABCDE 1 אופרון. </li><li style=";text-align:right;direction:rtl"> להכנה לממשל לעכברים, תרבויות הודגרו במדיום LB על 37 מעלות צלזיוס בייקרה ב 200 סל&quot;ד לשלב יומן האמצע-(צפיפות אופטית ב 600 ננומטר). חיידקים נקצרו ידי צנטריפוגה (6,000 × גרם למשך 5 דקות), נשטפו עם PBS (סיגמא), ודילול ב1 × PBS 10 7 יחידות מושבה להרכיב (CFU) / מ&quot;ל עבור IV ממשל, או 1 x 10 10 לgavage. </li></ol> 3. Administra קטריאליtion <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> עכברים חולקו באופן אקראי לקבוצות ניסוי, כאשר גידולים הגיעו לכ 100 מ&quot;מ 3 בנפח. עירוי לוריד, עכברים מאופקים כל אחד קבלו 10 6 תאים ב100 μl, מוזרקים ישירות לוריד הזנב לרוחב באמצעות מחט מזרק 28g. ספירת הקיימא של כל בידוד נקבעה על ידי ציפוי רטרוספקטיבית. </li><li style=";text-align:right;direction:rtl"> ללימודי קולוניזציה גית, 10 9 תאים חיידקיים היו אוראליים ב100 μl לעכבר על ידי gavage, על שלושה ימים רצופים. רמות חיידקים commensal קיים ירדו בלפני ההאכלה על ידי תוספת של סטרפטומיצין 5 מ&quot;ג / מ&quot;ל במי שתייה בעכבר למשך 7 ימים שקדם לתחילת gavage פי 1. </li></ol> 4. הדמית פליטת אור <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 2D in vivo BLI ההדמיה בוצע באמצעות IVIS100 (Caliper). בממשל הגדיר נקודתי זמן הודעת חיידקים, עכברים היו מורדמים באמצעות ההרדמה של Caliper XGI-8 הגזמערכת עם Isofluorane 3%, וניתוח תמונה של כל גוף בוצעה ב100 מערכת IVIS עבור 2-5 דקות ברגישות גבוהה. </li><li style=";text-align:right;direction:rtl"> להדמית 3D, עכברים מורדמים הונחו במעבורת הדמית עכבר הפנימי של מערכת ההדמיה האופטית להדמיה הגבה (IVIS ספקטרום, Caliper). לרכוש תמונות של אות לוציפראז חיידקים לשיקום אופטי 3D, אורכי גל נעים סינון פליטה 500-580 ננומטר שמשו עם 16 פעמי רכישת הסל של 3-4 דקות למסנן למקסם את יחס האות לרעש. כחלק מרצף רכישת התמונה, תמונת אור מובנהית הושגה להגדיר מפת גובה. מפה זה הייתה קלט מפוזרת אור הדמית אלגוריתמי שחזורים ששמשו ליצירת תמונה אופטית 3D באמצעות אופטימיזציה אינה שלילית לפחות ריבועי 2 טומוגרפיה (DLIT). </li><li style=";text-align:right;direction:rtl"> ניתוח תמונה: אזורים של עניין זוהה ולכמת באמצעות תוכנת תמונת חיים (Caliper). </li></ol> 5. נציגתוצאות במחקר זה, החיידקים פתוגניים commensal הלא E.coli K-12 MG1655 להביע luxABCDE אופרון היה רביעי ניתן לעכברי נושאי גידולי xenograft 4T1 sc. לוקס אות חיידקים זוהתה באופן ספציפי בגידולים של עכברי הודעת IV-ממשל (איור 2). התאוששות תרבות של חיידקים מעכברי מדגם מאמתת את קיומו של קשר לינארי בין מספרי חיידקי קיימא וכמות האור זוהה (איור 3). בתחום הדמית vivo של חיידקי commensal דרך פה המנוהל ב- Git גם מושג באמצעות 3D BLI. <img src="/files/ftp_upload/4318/4318fig1.jpg" alt="איור 1" fo:content-width="4in" fo:src="/files/ftp_upload/4318/4318fig1highres.jpg"/> איור 1. פרוטוקול ציר זמן. גידולים תת עורי הם המושרה בעכברים, וחיידקים מנוהלים על התפתחות גידול (100 מ&quot;מ 3). עכברים חיים הם BLI imהבן בממשל שונה בזמן נקודתי הודעת חיידקים (חיצים להציג זמנים טיפוסיים). <img src="/files/ftp_upload/4318/4318fig2.jpg" alt="איור 2" /> איור 2. מנהל של E. MG1655 luxABCDE coli לעכברי נושאים גידולים. גידולים תת עורי 4T1 היה מושרה בMF1 נו / נו עכברים וא MG1655 luxABCDE coli מנוהל על התפתחות גידולים. כל חיה קבלה 10 6 תאים הוזרקו ישירות לוריד הזנב לרוחב. עכברים צלמו בארבע נקודתי זמן במהלך המחקר (נקודות שחורות ציר Z-ותמונות) עם התאוששות מאוחרת של חיידקי קיימא (CFU) מגידולים של עכברים לדוגמה הקריבו (גרף עמודות). גידול במספרי חיידקים וביטוי גני פלסמיד במיוחד בגידולים נצפה לאורך זמן (עכבר נציג מאויר לכל נקודת זמן). <a href="http://www.jove.com/files/ftp_upload/4318/4318fig2large.jpg" target="_blank">לחץ כאן לצפייה בדמות גדולה.</a><img src="/files/ftp_upload/4318/4318fig3.jpg" alt="איור 3" /> איור 3. קשר בין מספרים חיידקיים intratumoral ופליטת אור. חיידקי קיימא בגידולים פורטו על ידי תרבות חיידקי vivo לשעבר מהגידולים הבאים לBLI בהודעת זמן נקודות שונים IV ממשל. היכנס ערכים של מספרי חיידקים (CFU) יחסית ליחידות vivo bioluminesce נמצאים בגרף. מתאם חזק בין ספירת חיידקים ואותות פליטת אור חיידקיים הוא ציין R 2 = 0.9717 1. <img src="/files/ftp_upload/4318/4318fig4.jpg" alt="איור 4" /> איור 4. תמונת 3D IVIS של מערכת העיכול Murine מאוכלסת על ידי א coli MG1655. Git של עכברים היה קולוניה אוראלית של 10 9 CFU של E. coli במשך שלושה ימים רצופים. דגימת תמונה בודדה מטומוגרפיה 3D של העכבר התנחל מוצגת.תמונות 3D להראות אטלס עכבר דיגיטלי של השלד לספק רישום אנטומי. א MG1655 פליטת אור coli גלויה בירוק בנמוך יותר, וסגולה ברמות גבוהות יותר.

<ol>
	<li>Cronin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+in+vivo+bioluminescent+imaging+for+the+study+of+bacterial+tumour+targeting.">High resolution in vivo bioluminescent imaging for the study of bacterial tumour targeting.</a> PLoS One. 7, e30940 (2012).</li><li>Kuo, C., Coquoz, O., Troy, T. L., Xu, H., Rice, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+reconstruction+of+in+vivo+bioluminescent+sources+based+on+multispectral+imaging.">Three-dimensional reconstruction of in vivo bioluminescent sources based on multispectral imaging.</a> J. Biomed. Opt. 12, 024007 (2007).</li><li>Tangney, M., Ahmad, S., Collins, S. A., O'Sullivan, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+for+prostate+cancer.">Gene therapy for prostate cancer.</a> Postgrad Med. 122, 166-180 (2010).</li><li>Morrissey, D., O'Sullivan, G. C., Tangney, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+targeting+with+systemically+administered+bacteria.">Tumour targeting with systemically administered bacteria.</a> Curr. Gene Ther. 10, 3-14 (2010).</li><li>Collins, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+vectors+in+cancer+immunotherapy:+which+vector+for+which+strategy.">Viral vectors in cancer immunotherapy: which vector for which strategy.</a> Curr. Gene Ther. 8, 66-78 (2008).</li><li>Yu, Y. A., Zhang, Q., Szalay, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+conditions+required+for+tumor+colonization+by+intravenously+delivered+bacteria.">Establishment and characterization of conditions required for tumor colonization by intravenously delivered bacteria.</a> Biotechnol. Bioeng. 100, 567-578 (2008).</li><li>Baban, C. K., Cronin, M., O'Hanlon, D., O'Sullivan, G. C., Tangney, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteria+as+vectors+for+gene+therapy+of+cancer.">Bacteria as vectors for gene therapy of cancer.</a> Bioeng. Bugs. 1, 385-394 (2010).</li><li>Cronin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orally+administered+bifidobacteria+as+vehicles+for+delivery+of+agents+to+systemic+tumors.">Orally administered bifidobacteria as vehicles for delivery of agents to systemic tumors.</a> Mol. Ther. 18, 1397-1407 (2010).</li><li>van Pijkeren, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+Listeria+monocytogenes-based+DNA+delivery+system+for+cancer+gene+therapy.">A novel Listeria monocytogenes-based DNA delivery system for cancer gene therapy.</a> Hum. Gene Ther. 21, 405-416 (2010).</li><li>Ahmad, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+effective+antitumor+response+after+mucosal+bacterial+vector+mediated+DNA+vaccination+with+endogenous+prostate+cancer+specific+antigen.">Induction of effective antitumor response after mucosal bacterial vector mediated DNA vaccination with endogenous prostate cancer specific antigen.</a> J. Urol. 186, 687-693 (2011).</li><li>Riedel, C. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+luciferase+tagging+system+for+Listeria+monocytogenes+allows+real-time+monitoring+in+vivo+and+in+vitro.">Improved luciferase tagging system for Listeria monocytogenes allows real-time monitoring in vivo and in vitro.</a> Appl Environ Microbiol. 73, 3091-3094 (2007).</li><li>Cheng, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-targeting+prodrug-activating+bacteria+for+cancer+therapy.">Tumor-targeting prodrug-activating bacteria for cancer therapy.</a> Cancer Gene Ther. 15, 393-401 (2008).</li><li>Foucault, M. L., Thomas, L., Goussard, S., Branchini, B. R., Grillot-Courvalin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+bioluminescence+imaging+for+the+study+of+intestinal+colonization+by+Escherichia+coli+in+mice.">In vivo bioluminescence imaging for the study of intestinal colonization by Escherichia coli in mice.</a> Appl. Environ. Microbiol. 76, 264-274 (2010).</li><li>Collins, S. A., Hiraoka, K., Inagaki, A., Kasahara, N., Tangney, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PET+Imaging+For+Gene+&amp;+Cell+Therapy.">PET Imaging For Gene &amp; Cell Therapy.</a> Curr. Gene Ther. , (2012).</li><li>Tangney, M., Francis, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Optical+Imaging+in+Gene+&amp;+Cell+Therapy.">In vivo Optical Imaging in Gene &amp; Cell Therapy.</a> Curr. Gene Ther. , (2012).</li></ol>

אין ניגודי האינטרסים הכריזו.

בהקשר של ריפוי גנטי, השימוש בחומרים ביולוגיים למסירה של גנים טיפוליים לחולים הוכיח הבטחה גדולה 3-5. כמו וירוסים, התכונות הביולוגיות המולדות של חיידקים מאפשרות אספקה ​​יעילה DNA לתאים או רקמות, בעיקר בהקשר של הסרטן. הוכח שחיידקים באופן טבעי מסוגלים ביות לגידולים,...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם המגיב </td><td> חברה </td><td> מספר קטלוגים </td><td> תגובות </td></tr><tr><td> שורת תאים 4T1 </td><td> ATCC </td><td> CRL-2539 </td><td> מודל סרטן השד Syngeneic נובע מגידול הנובע באופן ספונטני BALB / ג חלב </td></tr><tr><td> DMEM </td><td> סיגמה אולדריץ </td><td> D6429 </td><td> הבינוני של Dulbecco השינוי של הנשר </td></tr><tr><td> PBS </td><td> סיגמה אולדריץ </td><td> D8537 </td><td> נאגר מלוח פוספט </td></tr><tr><td> Xenogen IVIS </td><td> Caliper מדעי חיים </td><td></td><td> IVIS 100 עבור 2D הדמיה; ספקטרום IVIS 3D. </td></tr><tr><td> לוריא מרק מילר (LB) </td><td> סיגמה אולדריץ </td><td> L2542 </td><td> מדיום גידול עבור E. coli </td></p&gt; <tr><td> אריתרומיצין </td><td> סיגמה אולדריץ </td><td> E5389 </td><td> אנטיביוטי </td></tr><tr><td> סטרפטומיצין </td><td> סיגמה אולדריץ </td><td> S9137 </td><td> אנטיביוטי </td></tr><tr><td> עכברי MF1nu/nu </td><td> הרלן (בריטניה) </td><td> 069 (נו) / 070 (נו / +) </td><td> HSD: Athymic עירום-Foxn1nu </td></tr><tr><td> עכברי BALB / ג </td><td> הרלן (בריטניה) </td><td> 066 </td><td> Haplotype: H-2 ד </td></tr><tr><td> מחט Gavage </td><td> וטרינר טק פתרונות (בריטניה) </td><td> DE009 </td><td> 22g x 38mm מחט gavage ישר </td></tr><tr><td> מזרק להזרקת IV </td><td> BD Biosciences </td><td> 309309 - 1 מ&quot;ל </td><td> מזרק אינסולין עם 28-G ½ מחט אינץ x מייקרו הקנס הרביעי. </td></tr><tr><td> מזרק לחיסון גידול </td><td> בראון </td><td> 9161376V </td><td> Omnifix 26 x &amp; Gfrac12; מחט אינץ </td></tr></tbody></table>

bioluminescent bacterial imaging in vivo

וידאו זה מתאר את השימוש בגוף כולו bioluminesce ההדמיה (BLI) ללימודי סחר חיידקים בעכברים חיים, עם דגש על השימוש בחיידקים בגן וטיפול בתאי סרטן. חיידקים המצויים ברמה אטרקטיבית של וקטור לטיפול בסרטן, בעל יכולת טבעית לגדל מעדיף בתוך גידולים בעקבות מנהל מערכתי. חיידקים שהונדסו גנטי איתור לוקס קלטת היתר BLI של החיידק סרטניים ואתרים במקביל. המיקום ורמות חיידקים בתוך גידולים לאורך זמן, ניתן לבדוק בקלות, דמיין בשניים או שלושה ממדים. השיטה ישימה למגוון רחב של מינים וסוגי חיידקי xenograft סרטניים. מאמר זה מתאר את הפרוטוקול לניתוח של חיידקים בתוך bioluminescent עכברי נושאים גידולים תת עורי. ויזואליזציה של חיידקי commensal במערכת העיכול (git) על ידי BLI גם תארה. יציג זה חזק, וזול, בזמן אמת הדמית אסטרטגיהts שיטה אידיאלית לחקר חיידקים בחי בהקשר של חקר הסרטן, בטיפול בגן מסוים, ומחלות זיהומיות. סרטון זה מתאר את ההליך ללימוד ה-Lux מתויג coli בעכברים חיים, הוכחת BLI readout מרחב ובזמן להשגת ניצול עם מערכת IVIS.

Watch this Scientific Journal Video about Bioluminescent Bacterial Imaging In Vivo at JoVE.com

Bioluminescent Bacterial Imaging In Vivo

מאמר זה מתאר את הממשל של<em&gt; מתויג לוקס</em&gt; חיידקים לעכברים ולאחר מכן<em&gt; In vivo</em&gt; ניתוח באמצעות הדמית פליטת אור IVIS.

הדמיה בקטריאלי bioluminescent<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 ...

Research

JoVE Journal

Immunology and Infection

Bioluminescent Bacterial Imaging In Vivo

15.1K Views.  University College Cork. מאמר זה מתאר את הממשל של מתויג לוקס חיידקים לעכברים ולאחר מכן In vivo ניתוח באמצעות הדמית פליטת אור IVIS.

Video: Bioluminescent Bacterial Imaging In Vivo

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

בתחום ההדמיה vivo של טפילים טראנסגנטי שושנת יריחו שורה חיים

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

לא פולשני הדמיה של ביות ליקוציט והגירה In vivo</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

הערכת התגובה הפיוניות לחיות תאים חיידקיים באמצעות הדמיה עלה שלמים

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

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

באמצעות הדמיה Bioluminescent לחקור סינרגיזם בין Streptococcus pneumoniae ו שפעת וירוס עכברים תינוקות

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

&quot;Bioluminescent&quot; כתב phage לצורך זיהוי של קטגוריה פתוגנים חיידקיים

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

רוק, בלוטות הרוק, וכן אוסף Hemolymph מ קרציות קרצית Scapularis

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

איתור vivo מערכות הדמיה (IVIS) של וירוס אנצפליטים נוירו פולשנית

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

בשיטת ההדמיה vivo להבחין דלקת אקוטית וכרונית

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

דור והקרנה גבוהה תוכן רב-פנוטיפי של<em&gt; Burnetii Coxiella</em&gt; מוטאנטים טרנספוזון

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

במבחנת in vivo המודל ללמוד הידבקות חיידקים לכלי השיט וול תנאי זרימה תחת

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

הדמיה בקטריאלי bioluminescent

Summary

Explore More Videos

Chapters in this video