Name: imaging cell interaction in tracheal mucosa during influenza virus infection using two-photon intravital microscopy
Uploaded: 2018-08-17T15:00:00
Description: Watch this Scientific Journal Video about Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy at JoVE.com

Dette arbeidet ble støttet av den sveitsiske National Foundation (SNF) gir (176124, 145038 og 148183), den europeiske kommisjonen Marie Curie reintegrasjon Grant (612742) og SystemsX.ch for et stipend til D.U.P. (2013/124).

Alle dyr prosedyrer som involverer mus ble utført i henhold til sveitsiske føderale veterinær kontor retningslinjer og dyr protokollene ble godkjent av lokale veterinær myndighetene.
1. influensa infeksjon av CD11c-YFP mus
<ol><li>Biosikkerhet Merk: Mus tilpasset belastningen av influensa A/Puerto Rico/8/34 H1N1 (PR8) var vokst befruktede egg, renset og titreres som beskrevet tidligere21. Alle foranstaltningene innvolvere infiserte dyr eller bi...

<ol>
	<li>Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+two-photon+microscopy.">Deep tissue two-photon microscopy.</a> Nature Methods. 2 (12), 932-940 (2005).</li><li>Zipfel, W. R., Williams, R. M., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+magic:+multiphoton+microscopy+in+the+biosciences.">Nonlinear magic: multiphoton microscopy in the biosciences.</a> Nature biotechnology. 21 (11), 1369-1377 (2003).</li><li>Fein, M. R., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caught+in+the+act:+revealing+the+metastatic+process+by+live+imaging.">Caught in the act: revealing the metastatic process by live imaging.</a> Disease Models &amp; Mechanisms. 6 (3), 580-593 (2013).</li><li>Dombeck, D. A., Harvey, C. D., Tian, L., Looger, L. L., Tank, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+imaging+of+hippocampal+place+cells+at+cellular+resolution+during+virtual+navigation.">Functional imaging of hippocampal place cells at cellular resolution during virtual navigation.</a> Nature Neuroscience. 13 (11), 1433-1440 (2010).</li><li>Cahalan, M. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choreography+of+cell+motility+and+interaction+dynamics+imaged+by+two-photon+microscopy+in+lymphoid+organs.">Choreography of cell motility and interaction dynamics imaged by two-photon microscopy in lymphoid organs.</a> Annual review of immunology. 26, 585-626 (2008).</li><li>Germain, R. N., Robey, E. A., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Decade+of+Imaging+Cellular+Motility+and+Interaction+Dynamics+in+the+Immune+System.">A Decade of Imaging Cellular Motility and Interaction Dynamics in the Immune System.</a> Science. 336 (6089), 1676-1681 (2012).</li><li>Coombes, J. L., Robey, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+imaging+of+host-pathogen+interactions+in+vivo.">Dynamic imaging of host-pathogen interactions in vivo.</a> Nature Reviews Immunology. 10 (5), 353-364 (2010).</li><li>Pulendran, B., Maddur, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+Immune+Sensing+and+Response+to+Influenza.">Innate Immune Sensing and Response to Influenza.</a> Life Science Journal. 6 (4), 23-71 (2014).</li><li>Lim, K., 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=Neutrophil+trails+guide+influenza-+specific+CD8+++T+cells+in+the+airways.">Neutrophil trails guide influenza- specific CD8 + T cells in the airways.</a> Science. 349 (6252), (2015).</li><li>Kim, J. K., 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=In+vivo+imaging+of+tracheal+epithelial+cells+in+mice+during+airway+regeneration.">In vivo imaging of tracheal epithelial cells in mice during airway regeneration.</a> American journal of respiratory cell and molecular biology. 47 (6), 864-868 (2012).</li><li>Kretschmer, 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=Autofluorescence+multiphoton+microscopy+for+visualization+of+tissue+morphology+and+cellular+dynamics+in+murine+and+human+airways.">Autofluorescence multiphoton microscopy for visualization of tissue morphology and cellular dynamics in murine and human airways.</a> Laboratory investigation; a journal of technical methods and pathology. 96 (8), 918-931 (2016).</li><li>Veres, T. Z., 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=Intubation-free+in+vivo+imaging+of+the+tracheal+mucosa+using+two-photon+microscopy.">Intubation-free in vivo imaging of the tracheal mucosa using two-photon microscopy.</a> Scientific Reports. 7 (1), 694 (2017).</li><li>Looney, M. R., 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=Stabilized+imaging+of+immune+surveillance+in+the+mouse+lung.">Stabilized imaging of immune surveillance in the mouse lung.</a> Nature. 8 (1), 91-96 (2011).</li><li>Thornton, E. E., Krummel, M. F., Looney, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+of+the+Lung.">Live Imaging of the Lung.</a> Current Protocols in Cytometry. 60 (1), (2012).</li><li>Tabuchi, A., Mertens, M., Kuppe, H., Pries, A. R., Kuebler, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+of+the+murine+pulmonary+microcirculation.">Intravital microscopy of the murine pulmonary microcirculation.</a> Journal of Applied Physiology. 104 (2), 338-346 (2008).</li><li>Fiole, D., 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=Two-photon+intravital+imaging+of+lungs+during+anthrax+infection+reveals+long-lasting+macrophage-dendritic+cell+contacts.">Two-photon intravital imaging of lungs during anthrax infection reveals long-lasting macrophage-dendritic cell contacts.</a> Infection and immunity. 82 (2), 864-872 (2014).</li><li>Secklehner, J., Lo Celso, C., Carlin, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+in+historic+and+contemporary+immunology.">Intravital microscopy in historic and contemporary immunology.</a> Immunology and Cell Biology. 95 (6), 506-513 (2017).</li><li>Lambrecht, B. N., Hammad, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+Dendritic+Cells+in+Respiratory+Viral+Infection+and+Asthma:+From+Protection+to+Immunopathology.">Lung Dendritic Cells in Respiratory Viral Infection and Asthma: From Protection to Immunopathology.</a> Annual Review of Immunology. 30 (1), 243-270 (2012).</li><li>Camp, J. V., Jonsson, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+neutrophils+in+viral+respiratory+disease.">A role for neutrophils in viral respiratory disease.</a> Frontiers in Immunology. 8, (2017).</li><li>van Gisbergen, K. P. J. M., Sanchez-Hernandez, M., Geijtenbeek, T. B. H., van Kooyk, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+mediate+immune+modulation+of+dendritic+cells+through+glycosylation-dependent+interactions+between+Mac-1+and+DC-SIGN.">Neutrophils mediate immune modulation of dendritic cells through glycosylation-dependent interactions between Mac-1 and DC-SIGN.</a> The Journal of experimental medicine. 201 (8), 1281-1292 (2005).</li><li>Gonzalez, S. F., 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=Capture+of+influenza+by+medullary+dendritic+cells+via+SIGN-R1+is+essential+for+humoral+immunity+in+draining+lymph+nodes.">Capture of influenza by medullary dendritic cells via SIGN-R1 is essential for humoral immunity in draining lymph nodes.</a> Nature Immunology. 11 (5), 427-434 (2010).</li><li>Lindquist, R. L., 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=Visualizing+dendritic+cell+networks+in+vivo.">Visualizing dendritic cell networks in vivo.</a> Nature immunology. 5 (12), 1243-1250 (2004).</li><li>Li, H., 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=Human+V&#947;9V&#948;2-T+cells+efficiently+kill+influenza+virus-infected+lung+alveolar+epithelial+cells.">Human V&#947;9V&#948;2-T cells efficiently kill influenza virus-infected lung alveolar epithelial cells.</a> Cellular and Molecular Immunology. 10 (2), 159-164 (2013).</li><li>Tran Cao, H. 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=Development+of+the+transgenic+cyan+fluorescent+protein+(CFP)-expressing+nude+mouse+for+&amp;#34;technicolor&amp;#34;+cancer+imaging.">Development of the transgenic cyan fluorescent protein (CFP)-expressing nude mouse for &amp;#34;technicolor&amp;#34; cancer imaging.</a> Journal of Cellular Biochemistry. 107 (2), 328-334 (2009).</li><li>Jaber, S. 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=Dose+regimens,+variability,+and+complications+associated+with+using+repeat-bolus+dosing+to+extend+a+surgical+plane+of+anesthesia+in+laboratory+mice.">Dose regimens, variability, and complications associated with using repeat-bolus dosing to extend a surgical plane of anesthesia in laboratory mice.</a> Journal of the American Association for Laboratory Animal Science JAALAS. 53 (6), 684-691 (2014).</li><li>Pizzagalli, D. 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=Leukocyte+Tracking+Database,+a+collection+of+immune+cell+tracks+from+intravital+2-photon+microscopy+videos.">Leukocyte Tracking Database, a collection of immune cell tracks from intravital 2-photon microscopy videos.</a> Scientific Data. , (2018).</li><li>Sommer, C., Straehle, C., Kothe, U., Hamprecht, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ilastik:+Interactive+learning+and+segmentation+toolkit.">Ilastik: Interactive learning and segmentation toolkit.</a> 2011 IEEE International Symposium on Biomedical Imaging: From Nano to Macro. , 230-233 (2011).</li><li>Beltman, J. B., Mar&#233;e, A. F. M., De Boer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysing+immune+cell+migration.">Analysing immune cell migration.</a> Nature Reviews Immunology. 9 (11), 789-798 (2009).</li><li>Keller, H. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motility,+cell+shape,+and+locomotion+of+neutrophil+granulocytes.">Motility, cell shape, and locomotion of neutrophil granulocytes.</a> Cell motility. 3 (1), 47-60 (1983).</li><li>Sumen, C., Mempel, T. R., Mazo, I. B., von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+Microscopy.">Intravital Microscopy.</a> Immunity. 21 (3), 315-329 (2004).</li><li>Lambert Emo, K., 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=Live+Imaging+of+Influenza+Infection+of+the+Trachea+Reveals+Dynamic+Regulation+of+CD8++T+Cell+Motility+by+Antigen.">Live Imaging of Influenza Infection of the Trachea Reveals Dynamic Regulation of CD8+ T Cell Motility by Antigen.</a> PLOS Pathogens. 12 (9), e1005881 (2016).</li><li>Kjos, 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=Bright+fluorescent+Streptococcus+pneumoniae+for+live-cell+imaging+of+host-pathogen+interactions.">Bright fluorescent Streptococcus pneumoniae for live-cell imaging of host-pathogen interactions.</a> Journal of bacteriology. 197 (5), 807-818 (2015).</li></ol>

Dette arbeidet gir en detaljert protokoll for generering av 4D-bilder viser overføringen av adoptively overførte nøytrofile og deres samspill med fm under en influensa infeksjon i musen luftrøret. Beskrevet 2P-IVM modell vil være relevant å studere immun celle dynamics under en infeksjon i luftveiene.
Nylig har flere modeller basert på visualisering av cellen dynamikk i luftveiene vært utviklet9,10,...

To-fotonet intravital mikroskopi (2P-IVM) er en effektiv teknikk for sanntid tenkelig celle til celle samhandlinger som de forekommer i deres naturlige miljø1. En av de viktigste fordelene med denne metoden er at det tillater studiet av cellulære prosesser på en større prøven dybde (500 µm til 1 mm) sammenlignet med andre tradisjonelle imaging teknikker2. Samtidig minimerer bruk av to lav-energi fotoner generert av to-fotonet laser vev Foto-skade vanligvis assosiert med bilde oppkjøpet prosessen2. I løpet av det siste tiåret, er 2P-IVM brukt for å studere ulike typer celle-celle interaksjoner i flere disipliner3,4,5. Disse studiene har blitt spesielt relevant å undersøke immunceller, som er preget av deres høy dynamikk og dannelsen av fremtredende kontakter etter signalene generert av andre celler og miljø. 2P-IVM er også brukt for å studere interaksjonen mellom patogen og vert6. Faktisk har det tidligere vist at noen patogener kan endre type og varighet av kontaktene mellom immunceller, hindrer, resultatet av immunrespons7.
Luftveiene mucosa er det første stedet der immunrespons mot luftbårne patogener er generert8. I vivo analyse av patogen vert interaksjoner i dette vevet er derfor avgjørende å forstå initiering av forsvarsmekanismer vert under infeksjon. Men er 2P-IVM i luftveiene utfordrende skyldes gjenstander produsert av puste syklusen av dyret, som truer prosessen med bildeopptak. Nylig har ulike kirurgisk modeller blitt beskrevet for bildebehandling murine luftrøret9,10,11,12 og lungene13,14,15, 16. Tracheal 2P-IVM modeller representerer en utmerket oppsett å visualisere den innledende fasen av immunreaksjon i øvre luftveiene, mens lunge-alveoler 2P-IVM modeller er mer egnet til å studere den sene fasen av infeksjoner. Utviklet lunge modellene presentere en begrensning assosiert med tilstedeværelse av luftfylte alveoler, som begrenser optisk gjennomtrengning av laser og gjøre den slimhinnene lag i intrapulmonary luftveiene utilgjengelig for i vivo imaging17 . Omvendt, strukturen i luftrøret, dannet av en kontinuerlig epitel, forenkler bildeopptak.
Her presenterer vi en protokoll som inneholder en detaljert beskrivelse av trinn kreves for å utføre influensa infeksjon, kirurgisk utarbeidelse av dyrene, og 2P-IVM av trachea. I tillegg beskriver vi en bestemt eksperimentelle oppsett for visualisering av nøytrofile og dendrittiske celler (DC), to immun celletyper som spiller en viktig rolle som meglere på forsvar mekanisme mot influensa virus18,19 . Til slutt, vi beskriver en prosedyre for å analysere nøytrofile-DC interaksjoner. Kontaktene har vist seg å modulere DC aktivisering og, senere, å påvirke immunreaksjoner mot patogener20.

<table>
	<tbody>
		<tr>
			<td>Gigasept instru AF</td>
			<td>Sch&#252;lke &#38; Mayr GmbH</td>
			<td></td>
			<td>4% solution</td>
		</tr>
		<tr>
			<td>CD11c-YFP mice</td>
			<td>Jackson Laboratories</td>
			<td>008829</td>
			<td>mice were bred in-house</td>
		</tr>
		<tr>
			<td>CK6-ECFP mice</td>
			<td>Jackson Laboratories</td>
			<td>004218</td>
			<td>mice were bred in-house</td>
		</tr>
		<tr>
			<td>1 X Dulbecco&#39;s Phosphate Buffered Saline modified without Calcium Choride and Magnesium Chloride</td>
			<td>Sigma</td>
			<td>D8537-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>10 X Dulbecco&#39;s Phosphate Buffered Saline modified without Calcium Choride and Magnesium Chloride</td>
			<td>Sigma</td>
			<td>D1408-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll PLUS</td>
			<td>Sigma</td>
			<td>E0414-1L</td>
			<td>Store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Ketamin Labatec</td>
			<td>Labatec Pharma</td>
			<td>7680632310024</td>
			<td>Store at RT, store at 4&#176;C when in solution of ket/xyl mixture</td>
		</tr>
		<tr>
			<td>Rompun 2% (Xylazin)</td>
			<td>Bayer</td>
			<td>6293841.00.00</td>
			<td>Store at RT, store at 4&#176;C when in solution of ket/xyl mixture</td>
		</tr>
		<tr>
			<td>26 G 1 mL Sub-Q BD Plastipak</td>
			<td>BD Plastipak</td>
			<td>305501</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G 0,3 mL BD Micro-Fine Insulin Syringes</td>
			<td>BD</td>
			<td>324826</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 40 &#181;m Cell Strainer</td>
			<td>Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Syringes</td>
			<td>BD Plastipak</td>
			<td>300185</td>
			<td></td>
		</tr>
		<tr>
			<td>Microlance 3 18 G needles</td>
			<td>BD</td>
			<td>304622</td>
			<td></td>
		</tr>
		<tr>
			<td>Introcan Safety 20G (catheter)</td>
			<td>Braun</td>
			<td>4251652.01</td>
			<td></td>
		</tr>
		<tr>
			<td>6 Well Cell Culture Cluster</td>
			<td>Costar</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium 1640 + HEPES (1X)</td>
			<td>ThermoFisher Scientific</td>
			<td>42401-018</td>
			<td>Store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Liberase TL Research Grade</td>
			<td>Roche</td>
			<td>5401020001</td>
			<td>Store at -20&#176;C / collagenase (I and II) mixture</td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Amresco (VWR)</td>
			<td>0649-50KU</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td>CellTrace Violet stain</td>
			<td>ThermoFisher Scientific</td>
			<td>C34557</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>EDS-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td>PE-10 Micro Medical Tubing</td>
			<td>2Biological Instruments SNC</td>
			<td>#BB31695-PE/1</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Plastic Tape</td>
			<td>M Plast</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Viscotears</td>
			<td>Bausch &#38; Lomb</td>
			<td></td>
			<td>Store at RT</td>
		</tr>
		<tr>
			<td>Plasticine</td>
			<td>Ohropax</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High Tolerance Glass Coverslip 15mm Round</td>
			<td>Warner Instruments</td>
			<td>64-0733</td>
			<td></td>
		</tr>
		<tr>
			<td>SomnoSuite Portable Animal Anesthesia System</td>
			<td>Kent Scientific</td>
			<td>SS-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuvo Lite mark 5</td>
			<td>GCE medline</td>
			<td>14111211</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniTag (gaseous anesthesia and heating bench)</td>
			<td>Tem Sega</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SURGICAL BOARD</td>
			<td>University of Bern</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TrimScope II Two-photon microscope</td>
			<td>LaVision Biotec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chameleon Vision Ti:Sa lasers</td>
			<td>Coherent Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25X NA 1.05 water immersion objective</td>
			<td>Olympus</td>
			<td>XLPLN25XWMP2</td>
			<td></td>
		</tr>
		<tr>
			<td>The Cube&#38;The Box incubation chamber and temperature controller</td>
			<td>Life imaging Services</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris 9.1.0</td>
			<td>Bitplane</td>
			<td></td>
			<td>Imaging software</td>
		</tr>
		<tr>
			<td>GraphPad Prism 7</td>
			<td>GraphPad</td>
			<td></td>
			<td>Statistical software</td>
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	</tbody>
</table>

imaging cell interaction in tracheal mucosa during influenza virus infection using two-photon intravital microscopy

Analyse av celle-celle eller cellen-patogen samhandling i vivo er et viktig verktøy for å forstå dynamikken i immun respons på infeksjon. To-fotonet intravital mikroskopi (2P-IVM) gir observasjon av cellen interaksjoner i dype vev i levende dyr, samtidig som photobleaching genereres under bildeopptak. Ulike modeller for 2P-IVM av lymfoide og ikke-lymfoide organer er beskrevet hittil. Bildebehandling åndedretts organer er imidlertid en utfordring på grunn av bevegelsen knyttet pust syklusen av dyret.
Her beskriver vi en protokoll for å visualisere i vivo immun celle interaksjoner i luftrøret mus infisert med influensavirus benytter 2 P-IVM. Til dette formålet utviklet vi en egendefinert tenkelig plattform, som inkluderte kirurgisk eksponering og intubasjon av trachea, etterfulgt av oppkjøpet av dynamiske bilder av nøytrofile og dendrittiske celler (DC) i slimhinnene epitel. I tillegg detaljerte vi trinnene for å utføre influensa intranasal infeksjon og flyt cytometric analyse av immunceller i luftrøret. Til slutt, vi analyserte nøytrofile og DC motilitet samt deres samspill i løpet av en film. Denne protokollen tillater generering av stabile og lyse 4D-bilder nødvendig for å vurdere celle-celle interaksjoner i luftrøret.

I dette arbeidet beskrev vi en detaljert protokoll for å studere i vivo motilitet og samspillet mellom nøytrofile og DC under influensa infeksjon i murine luftrøret (figur 3A). Til dette formålet isolerte vi CFP+ nøytrofile (92% renhet; Figur 3B) fra CK6-ECFP overført mus og vi adoptively dem til en CD11c-YFP mus infisert med influensa. Etter at spilte vi 2P-IVM av trachea på dag 3 p.i. På dette tidspu...

Watch this Scientific Journal Video about Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy at JoVE.com

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

I denne studien presenterer vi en protokoll for å utføre to-fotonet intravital imaging og celle samhandling analyse i murine tracheal mucosa etter infeksjon med influensavirus. Denne protokollen vil være relevant for forskere studere immun celle dynamics under luftveisinfeksjoner.

Bildebehandling celle interaksjon i Tracheal Mucosa under influensa Virus smitte med to-fotonet Intravital mikroskopi

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

Research

JoVE Journal

Immunology and Infection

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

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 ...

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Long Term intra multiphoton Mikros Imaging av immunceller i friske og syke leveren Bruke CXCR6.Gfp Reporter Mus

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Nøytrofile Isolering og analyse for å fastslå deres rolle i lymfom Cell Følsomhet for Terapeutiske midler

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

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Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

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Cellemembranen reparasjon analysen ved hjelp av to-fotonet Laser mikroskop

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

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

To-fotonet Intravital Imaging av leukocytter under immunrespons i lipopolysakkarid-behandlet musen leveren

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

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Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

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Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, ...