Name: extraction of hemocytes from drosophila melanogaster larvae for microbial infection and analysis
Uploaded: 2018-05-24T13:00:00
Description: Watch this Scientific Journal Video about Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis at JoVE.com

Wir sind dankbar, Dr. Robert Heinzen für die Bereitstellung von Aktien von mCherry exprimierenden Coxiella Burnetii. Wir danken Dr. Luis Teixeira für Bereitstellung von Wirbellosen irisierende Virus 6 und Bloomington Stock Center für die Bereitstellung fliegen Bestände. Dieses Projekt wurde teilweise von NIH Grant R00 AI106963 (A.G.G) und der Washington State University finanziert.

1. ex Vivo -Infektion
<ol><li>Medium und Ausrüstung<ol>
<li>Unter sterilen Bedingungen bereiten frische Drosophila Hemocyte isolieren Medium (DHIM) mit 75 % Schneider Drosophila mit 25 % mittlere fetalen Bovine Serum (FBS) und Filter Sterilisieren sie.</li>
		<li>Layer-2-3 Stücke von 10 x 10 cm Paraffin Film unter einem Stereomikroskop.</li>
		<li>Bereiten Sie die Glas-Kapillare. Legen Sie die Kapillare Puller-Heizung auf 55 % des Maximums. Ziehen Sie das Kapillarrohr zu einer scharfen Spit...

<ol>
	<li>Hoffmann, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immune+response+of+Drosophila.">The immune response of Drosophila.</a> Nature. 426 (6962), 33-38 (2003).</li><li>Regan, J. C., 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=Steroid+hormone+signaling+is+essential+to+regulate+innate+immune+cells+and+fight+bacterial+infection+in+Drosophila.">Steroid hormone signaling is essential to regulate innate immune cells and fight bacterial infection in Drosophila.</a> PLoS Pathog. 9 (10), 1003720 (2013).</li><li>Yano, T., 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=Autophagic+control+of+listeria+through+intracellular+innate+immune+recognition+in+Drosophila.">Autophagic control of listeria through intracellular innate immune recognition in Drosophila.</a> Nat Immunol. 9 (8), 908-916 (2008).</li><li>Bastos, R. G., Howard, Z. P., Hiroyasu, A., Goodman, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+and+Bacterial+Factors+Control+Susceptibility+of+Drosophila+melanogaster+to+Coxiella+burnetii+Infection.">Host and Bacterial Factors Control Susceptibility of Drosophila melanogaster to Coxiella burnetii Infection.</a> Infect Immun. 85 (7), (2017).</li><li>Kacsoh, B. Z., Schlenke, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+hemocyte+load+is+associated+with+increased+resistance+against+parasitoids+in+Drosophila+suzukii,+a+relative+of+D.+melanogaster.">High hemocyte load is associated with increased resistance against parasitoids in Drosophila suzukii, a relative of D. melanogaster.</a> PLoS One. 7 (4), 34721 (2012).</li><li>Tsuzuki, 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=Switching+between+humoral+and+cellular+immune+responses+in+Drosophila.+is+guided+by+the+cytokine+GBP.">Switching between humoral and cellular immune responses in Drosophila. is guided by the cytokine GBP.</a> Nat Commun. 5, 4628 (2014).</li><li>Kurucz, E., 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=Definition+of+Drosophila.+hemocyte+subsets+by+cell-type+specific+antigens.">Definition of Drosophila. hemocyte subsets by cell-type specific antigens.</a> Acta Biol Hung. 58, 95-111 (2007).</li><li>Neyen, C., Bretscher, A. J., Binggeli, O., Lemaitre, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+study+Drosophila+immunity.">Methods to study Drosophila immunity.</a> Methods. 68 (1), 116-128 (2014).</li><li>Arefin, B., 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=Apoptosis+in+Hemocytes+Induces+a+Shift+in+Effector+Mechanisms+in+the+Drosophila.+Immune+System+and+Leads+to+a+Pro-Inflammatory+State.">Apoptosis in Hemocytes Induces a Shift in Effector Mechanisms in the Drosophila. Immune System and Leads to a Pro-Inflammatory State.</a> PLoS One. 10 (8), 0136593 (2015).</li><li>Rus, 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=Expression+pattern+of+Filamin-240+in+Drosophila+blood+cells.">Expression pattern of Filamin-240 in Drosophila blood cells.</a> Gene Expr Patterns. 6 (8), 928-934 (2006).</li><li>Kurucz, E., 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=Hemese,+a+hemocyte-specific+transmembrane+protein,+affects+the+cellular+immune+response+in+Drosophila.">Hemese, a hemocyte-specific transmembrane protein, affects the cellular immune response in Drosophila.</a> P Natl Acad Sci USA. 100 (5), 2622-2627 (2003).</li><li>Markus, 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=Sessile+hemocytes+as+a+hematopoietic+compartment+in+Drosophila+melanogaster.">Sessile hemocytes as a hematopoietic compartment in Drosophila melanogaster.</a> P Natl Acad Sci USA. 106 (12), 4805-4809 (2009).</li><li>Bretscher, A. J., 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=The+Nimrod+transmembrane+receptor+Eater+is+required+for+hemocyte+attachment+to+the+sessile+compartment+in+Drosophila+melanogaster.">The Nimrod transmembrane receptor Eater is required for hemocyte attachment to the sessile compartment in Drosophila melanogaster.</a> Biol Open. 4 (3), 355-363 (2015).</li><li>Zettervall, C. J., 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+directed+screen+for+genes+involved+in+Drosophila+blood+cell+activation.">A directed screen for genes involved in Drosophila blood cell activation.</a> P Natl Acad Sci USA. 101 (39), 14192-14197 (2004).</li><li>Flynt, A., Liu, N., Martin, R., Lai, E. 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T., Eleftherianos, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+protocol+for+extracting+hemocytes+from+wild+caterpillars.">A simple protocol for extracting hemocytes from wild caterpillars.</a> J Vis Exp. (69), e4173 (2012).</li><li>Sampson, C. J., Williams, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+ex+vivo+incubation+of+Drosophila+primary+post-embryonic+haemocytes+for+real-time+analyses.">Protocol for ex vivo incubation of Drosophila primary post-embryonic haemocytes for real-time analyses.</a> Methods Mol Biol. 827, 359-367 (2012).</li><li>Nehme, N. T., 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+model+of+bacterial+intestinal+infections+in+Drosophila+melanogaster.">A model of bacterial intestinal infections in Drosophila melanogaster.</a> PLoS Pathog. 3 (11), 173 (2007).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+fruit+juice+egg+plates.">Drosophila fruit juice egg plates.</a> Cold Spring Harbor Protocols. (9), (2007).</li><li>Ahlers, L. R., Bastos, R. G., Hiroyasu, A., Goodman, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invertebrate+Iridescent+Virus+6,+a+DNA+Virus,+Stimulates+a+Mammalian+Innate+Immune+Response+through+RIG-I-Like+Receptors.">Invertebrate Iridescent Virus 6, a DNA Virus, Stimulates a Mammalian Innate Immune Response through RIG-I-Like Receptors.</a> PLoS One. 11 (11), 0166088 (2016).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments.">Agarose gel electrophoresis for the separation of DNA fragments.</a> J Vis Exp. (62), (2012).</li><li>Petraki, S., Alexander, B., Bruckner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+Blood+Cell+Populations+of+the+Drosophila+melanogaster+Larva.">Assaying Blood Cell Populations of the Drosophila melanogaster Larva.</a> J Vis Exp. (105), (2015).</li><li>Figliozzi, R. W., Chen, F., Chi, A., Hsia, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+inverse+Poisson+distribution+to+calculate+multiplicity+of+infection+and+viral+replication+by+a+high-throughput+fluorescent+imaging+system.">Using the inverse Poisson distribution to calculate multiplicity of infection and viral replication by a high-throughput fluorescent imaging system.</a> Virol Sin. 31 (2), 180-183 (2016).</li><li>Rizki, T. M., Rizki, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lamellocyte+differentiation+in+Drosophila.+larvae+parasitized+by+Leptopilina.">Lamellocyte differentiation in Drosophila. larvae parasitized by Leptopilina.</a> Dev Comp Immunol. 16 (2-3), 103-110 (1992).</li><li>Markus, R., Kurucz, E., Rus, F., Ando, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sterile+wounding+is+a+minimal+and+sufficient+trigger+for+a+cellular+immune+response+in+Drosophila+melanogaster.">Sterile wounding is a minimal and sufficient trigger for a cellular immune response in Drosophila melanogaster.</a> Immunol Lett. 101 (1), 108-111 (2005).</li><li>McCormack, 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=Perforin-2+Protects+Host+Cells+and+Mice+by+Restricting+the+Vacuole+to+Cytosol+Transitioning+of+a+Bacterial+Pathogen.">Perforin-2 Protects Host Cells and Mice by Restricting the Vacuole to Cytosol Transitioning of a Bacterial Pathogen.</a> Infect Immun. 84 (4), 1083-1091 (2016).</li><li>Ozgen, 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=Construction+and+characterization+of+a+recombinant+invertebrate+iridovirus.">Construction and characterization of a recombinant invertebrate iridovirus.</a> Virus Res. 189, 286-292 (2014).</li><li>Jakob, N. J., Muller, K., Bahr, U., Darai, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+first+complete+DNA+sequence+of+an+invertebrate+iridovirus:+coding+strategy+of+the+genome+of+Chilo+iridescent+virus.">Analysis of the first complete DNA sequence of an invertebrate iridovirus: coding strategy of the genome of Chilo iridescent virus.</a> Virology. 286 (1), 182-196 (2001).</li><li>Ghigo, E., Colombo, M. I., Heinzen, R. 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Um besser zu verstehen, wie Wirtszellen infiziert werden, ist es wichtig, die Lokalisation des Erregers in den Zellen zu klären, vor allem, wenn zuvor noch nicht getesteten Erreger und Zelle Typ Kombinationen4experimentieren. Während Studium der zellulären Antwort Kaskade nach Infektion produktive Erreger Invasion anzeigen kann, gilt die Kombination von bildgebenden und zellulären Antwortdaten Erreger Invasion und Infektion nachweisen. Während Berichte zeigen 2D-Bilder Erreger Invasion in die...

Drosophila Melanogaster ist eine gut etablierte Modellorganismus zur Erforschung der angeborenen Immunität1. Im Rahmen der angeborenen Immunantwort spielen Hemocytes eine wichtige Rolle bei der Reaktion auf Krankheitserreger Herausforderung. Hemocytes sind entscheidend für die Kapselung von Parasiten, sowie eine wichtige Funktion bei der Bekämpfung des Erregers durch phagocytic Aktion während Pilz-, Virus- und bakterielle Infektion2,3.
Um den Host angeborene Immunantwort auf pathogene mikrobielle Infektion am besten zu verstehen, ist es wichtig, wie der Erreger dringt in Wirtszellen während der Infektion zu visualisieren. Diese Visualisierung trägt zum Verständnis des Mechanismus der Invasion. Zusammen mit Details der Erreger intrazelluläre Lokalisation und die zelluläre Antwort können diese Daten Aufschluss über der Wirtsantwort zur Infektion und die zellularen Organellen die Mikrobe interagiert. 3D-Modell Wiederaufbau nach Bildgebung durch Mikroskopie kann hilfreich, um die genaue Lage von Krankheitserregern in Wirtszellen zu bestimmen. In dieser Studie visualisieren wir die Invasion von Coxiella Burnetii (C. Burnetii), dem Erreger der q-Fieber, eine Zoonose, die eine ernsthafte für die menschliche und tierische Gesundheit in primären Drosophila Hemocytes Bedrohung. Vor kurzem wurde gezeigt, dass Drosophila sind anfällig für die Sicherheitsstufe 2 Nine Mile Phase II (NMII) Klon 4 Stamm von C. Burnetii und, dass diese Sorte in Drosophila4replizieren kann darauf hinweist, dass Drosophila einsetzbar als Modellorganismus C. Burnetii Pathogenese zu studieren.
Frühere Studien haben Hemocytes verwendet, um angeborene Immunantwort des Wirtes zu untersuchen. Hemocytes wurden verwendet für morphologische Beobachtungen5,6,7, morphometrische Analyse2,8, Phagozytose Analyse2,3, qRT-PCR-2 , 9, Immunopräzipitation10,11, immunofluorescent Analyse10,12, Immunostaining13, Immunoblotting3,10, 11 und Immunohistochemistry9,14. Obwohl Drosophila S2 Zellen auch für verschiedene in-vitro- Experimente zur Verfügung stehen, ändern Immortalisierung und potenzielle bereits bestehende Virusinfektion ihr Verhalten15,16. Die Verwendung von Primärzellen im Gegensatz zu einer immortalisierte Zelllinie wie S2-Zellen, kann für das Studium der angeborene immune Funktion in einem System repräsentativer für den gesamten Organismus. Darüber hinaus kann die Infektion von Hemocytes in Vivo, vor der Extraktion, die Zellen, die Interaktion mit anderen Host Proteine und Gewebe, einen Vorteil gegenüber der Gewinnung von Hemocytes vor der ex-Vivo -Infektion. Eine Reihe von verschiedenen Methoden wurden genutzt, um eine ausreichende Anzahl von Hemocytes in kurzer Zeit zu den Hemocytes lebendig8,17,18,19zu erhalten.
In dieser Studie stellen wir Ihnen eine Methode zur Hemocytes von Drosophila 3rd Instar Larven für pathogene mikrobielle Infektion mit C. Burnetii, Listeria Monocytogenes (Listerien) oder Wirbellosen schillernden extrahieren Virus 6 (IIV6). Wir beschreiben die Methoden zur in Vivo und ex Vivo Hemocyte Infektionen. In Vivound ex Vivo-infizierten Hemocytes waren mit der konfokalen Mikroskopie visualisiert und verwendet zum Erstellen von 3D Modellen von C. Burnetii Invasion. Darüber hinaus verwenden die Extraktion Protokoll, ex-Vivo-infizierten Hemocytes dienten für gen- und Protein-Expression Assays. Insbesondere war um zu prüfen, inwieweit der Infektion mit IIV6 und Listeria, total RNA oder Protein isoliert aus den Zellen zur qRT-PCR oder Western-Blot Analyse. Zusammen genommen, das Protokoll bietet Methoden, um schnell hohe Zahl von Hemocytes von 3rd Instar Larven und Beweise, dass primäre Hemocytes infiziert, in Vivo und Ex Vivosammeln, sind eine geeignete Plattform für mikrobielle Erreger Infektion Studien und anwendbaren nachgeschalteten Analysen wie Mikroskopie, Transkriptom und Proteomik.

<table border="0" cellpadding="0" cellspacing="0" style="width:1128px;" width="1125">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:303px;">Schneider&#39;s Drosophila Medium&#160;</td>
			<td style="width:288px;">Thermo Fisher Scientific (Gibco)</td>
			<td style="width:145px;">21720024</td>
			<td style="width:179px;">1.1.1), 2.1.2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Fetal Bovine Serum</td>
			<td style="width:288px;">GE Healthcare Life Sciences&#160; (HyClone)</td>
			<td style="width:145px;">SH30070.03HI</td>
			<td style="width:179px;">1.1.1), 2.1.2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Filter (0.22 &#181;L)</td>
			<td style="width:288px;">RESTEK</td>
			<td style="width:145px;">26158</td>
			<td style="width:179px;">1.1.1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Strainer (100 &#181;m)</td>
			<td style="width:288px;">Greiner bio-one</td>
			<td style="width:145px;">542000</td>
			<td>1.2.1), 2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Stereo microscope</td>
			<td style="width:288px;">Amscope</td>
			<td style="width:145px;">SM-1BSZ-L6W</td>
			<td style="width:179px;">1.2), 2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Glass capillary</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:145px;">21-171-4</td>
			<td style="width:179px;">1.1), 1.2), 2)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Capillary puller</td>
			<td style="width:288px;">Narishige International USA, Inc.</td>
			<td style="width:145px;">PC-10</td>
			<td style="width:179px;">1.1.3)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Mineral oil</td>
			<td style="width:288px;">Snow River Products</td>
			<td style="width:145px;"></td>
			<td style="width:179px;">1.1.4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Nanoinjector</td>
			<td style="width:288px;">Drummond Scientific Company</td>
			<td style="width:145px;">3-000-204</td>
			<td style="width:179px;">1.1), 1.2), 2.2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Forceps</td>
			<td style="width:288px;">VWR</td>
			<td style="width:145px;">82027-402</td>
			<td style="width:179px;">1.1.5), 1.2), 2), 3.1.7)</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:303px;">CO2 delivery apparatus</td>
			<td style="width:288px;">Genesee Scientific</td>
			<td style="width:145px;">59-122BC</td>
			<td style="width:179px;">1.2), 2)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Trypan Blue</td>
			<td style="width:288px;">Thermo Fisher Scientific (Gibco)</td>
			<td style="width:145px;">15250061</td>
			<td style="width:179px;">1.3)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Hemocytometer</td>
			<td style="width:288px;">Hausser Scientific</td>
			<td style="width:145px;">3100</td>
			<td style="width:179px;">1.3)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">24 well plate</td>
			<td style="width:288px;">Greiner bio-one</td>
			<td style="width:145px;">662160</td>
			<td style="width:179px;">1.4), 2.2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Coxiella burnetii - mCherry</td>
			<td style="width:288px;">Dr. Heinzen, R.</td>
			<td style="width:145px;"></td>
			<td style="width:179px;">1.4), 2.2)</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:303px;">Drosophila fruit juice plates</td>
			<td style="width:288px;">Cold Spring Harbor Protocols</td>
			<td style="width:145px;"></td>
			<td>2.1) http://cshprotocols.cshlp.org/content/2007/9/pdb.rec11113.full</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Agar</td>
			<td style="width:288px;">Fisher Bioreagents</td>
			<td style="width:145px;">BP1423-500</td>
			<td style="width:179px;">2.1.1.1)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Methyl paraben</td>
			<td style="width:288px;">Amresco</td>
			<td style="width:145px;">0572-500G</td>
			<td style="width:179px;">2.1.1.2)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Absolute ethanol</td>
			<td style="width:288px;">Fisher Bioreagents</td>
			<td style="width:145px;">BP2818-500</td>
			<td style="width:179px;">2.1.1.2)</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:303px;">Welch&#39;s 100% Grape juice frozen concentrate, 340 mL</td>
			<td style="width:288px;">Amazon</td>
			<td style="width:145px;">B0025UJVGM</td>
			<td style="width:179px;">2.1.1.3)</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:303px;">Petri dishes, 10 x 35 mm</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:145px;">08-757-100A</td>
			<td style="width:179px;">2.1.1.4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Microscope cover glass</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:145px;">12-545-80</td>
			<td style="width:179px;">1.4.4), 2.2.2)</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:303px;">Yeast, Bakers Dried Active</td>
			<td style="width:288px;">MP Biomedicals</td>
			<td style="width:145px;">0210140001</td>
			<td>2.1) Add 2 parts of water to 1 part of yeast (v/v)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Tungsten needle</td>
			<td style="width:288px;">Fine Science Tools</td>
			<td style="width:145px;">10130-20</td>
			<td style="width:179px;">2.1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Holding forceps</td>
			<td style="width:288px;">VWR</td>
			<td style="width:145px;">HS8313</td>
			<td style="width:179px;">2.1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Paraformaldehyde</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:145px;">FLO4042-500</td>
			<td style="width:179px;">3.1.3)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Triton X-100</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:145px;">BP151-500</td>
			<td style="width:179px;">3.1.3)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Bovine Serum Albumin</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:145px;">BP9706-100</td>
			<td style="width:179px;">3.1.3)</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:303px;">4&#39;,6-diamidino-2-phenylindole</td>
			<td style="width:288px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">62247</td>
			<td style="width:179px;">3.1.4)</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:303px;">Antifade mounting medium</td>
			<td style="width:288px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">P36930</td>
			<td style="width:179px;">3.1.6)</td>
		</tr>
		<tr height="132">
			<td height="132" style="height:132px;width:303px;">Confocal microsope</td>
			<td style="width:288px;">Leica</td>
			<td style="width:145px;">TCS SP8-X White Light Confocal Laser Scanning Microscope</td>
			<td style="width:179px;">3.2)</td>
		</tr>
		<tr height="103">
			<td height="103" style="height:103px;width:303px;">3D imaging reconstruction software</td>
			<td style="width:288px;">Leica</td>
			<td style="width:145px;">LASX with 3D visualization module</td>
			<td style="width:179px;">3.3)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Microscope slides</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:145px;">12-552-3</td>
			<td style="width:179px;">3.1.6)</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:303px;">Invertebrate iridescent virus 6 (IIV6)</td>
			<td style="width:288px;">Dr. Teixeria, L.</td>
			<td style="width:145px;"></td>
			<td>4) PLoS Biol, 6 (12), 2753-2763, doi: 10.1371/journal.pbio.1000002, (2008)</td>
		</tr>
		<tr height="141">
			<td height="141" style="height:141px;width:303px;">Listeria monocytogenes</td>
			<td style="width:288px;">ATCC</td>
			<td style="width:145px;">strain: 10403S</td>
			<td>4) Listeria monocytogenes strain 10403S (Bishop and Hinrichs, 1987) was grown in Difco Brain-heart infusion (BHI) broth (BD Biosciences) containing 50 &#181;g/ml streptomycin at 30 &#176;C.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">DNase I</td>
			<td style="width:288px;">Thermo Fisher Scientific(Invitrogen)</td>
			<td style="width:145px;">18068015</td>
			<td>gDNA degradation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">cDNA Synthesis Kit</td>
			<td style="width:288px;">Bio-Rad</td>
			<td style="width:145px;">1708891</td>
			<td>cDNA synthesis</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">IIV6_193R_F</td>
			<td style="width:288px;">IDT</td>
			<td style="width:145px;"></td>
			<td>qRT-PCR, 5&#39;- TCT TGT TTT CAG AAC CCC ATT -3&#39;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">IIV6_193R_R</td>
			<td style="width:288px;">IDT</td>
			<td style="width:145px;"></td>
			<td>qRT-PCR, 5&#39;- CAC GAA GAA TGA CCA CAA GG -3&#39;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">RpII_qRTPCR_fwd</td>
			<td style="width:288px;">SIGMA-ALDRICH</td>
			<td style="width:145px;"></td>
			<td>qRT-PCR, 5&#39;- GAA GCG TTT CTC CAA ACG -AG</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">RpII_qRTPCR_rev</td>
			<td style="width:288px;">SIGMA-ALDRICH</td>
			<td style="width:145px;"></td>
			<td>qRT-PCR, 5&#39;- TTG AGC GTA AGC ATC ACC -TG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">SYBR Green qRT-PCR reagent</td>
			<td style="width:288px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">K0251, K0252, K0253</td>
			<td>qRT-PCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Real-Time PCR System</td>
			<td style="width:288px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">4351107, 7500 Software v2.0</td>
			<td>qRT-PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Anti-Listeria monocytogenes antibody</td>
			<td style="width:288px;">abcam</td>
			<td style="width:145px;">ab35132</td>
			<td>Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Anti-Actin antibody produced in rabbit</td>
			<td style="width:288px;">SIGMA-ALDRICH</td>
			<td style="width:145px;">A2066</td>
			<td>Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Anti-Rabbit IgG (H+L), HRP Conjugate</td>
			<td style="width:288px;">Promega</td>
			<td style="width:145px;">W4011</td>
			<td>Western blot</td>
		</tr>
	</tbody>
</table>

extraction of hemocytes from drosophila melanogaster larvae for microbial infection and analysis

Während die pathogenen Infektion von Drosophila Melanogaster, Rolle Hemocytes eine wichtige bei der Immunantwort in die Infektion. Ziel dieses Protokolls ist es daher, Entwicklung eine Methode um die Invasion der Erreger in einem bestimmten immun Fach fliegen, nämlich Hemocytes zu visualisieren. Mit der Methode, die hier vorgestellten, bis zu 3 × 106 live Hemocytes von 200 Drosophila 3rd Instar Larven in 30 min für Ex-Vivo -Infektion erhalten. Alternativ kann die Hemocytes infizierte in Vivo durch Injektion von 3rd Instar Larven gefolgt von Hemocyte Extraktion bis zu 24 h nach der Infektion. Diese infizierten Primärzellen gefixt, gebeizt und abgebildet mit der konfokalen Mikroskopie. Dann wurden aus den Bildern endgültig Erreger Invasion zeigen 3D-Darstellungen generiert. Darüber hinaus hochwertige RNA für die qRT-PCR für den Nachweis des Erregers mRNA folgenden erhalten werden Infektionen und ausreichend Protein aus diesen Zellen für Western-Blot Analyse extrahiert werden können. Zusammengenommen, präsentieren wir Ihnen eine Methode zur definitiven Versöhnung der Erreger Invasion und Bestätigung der Infektion mit bakterieller und viraler Erreger-Typen und eine effiziente Methode für die Extraktion von Hemocyte genug live Hemocytes von Drosophila zu erhalten Larven für ex-Vivo - und in-Vivo -Infektions-Experimente.

Zu sammeln Hemocytes für Ex-Vivo -Infektion, Leben bis zu 3 × 10 wurden 200 Drosophila 3rd Instar Larven6 Hemocytes entzogen. Um unsere Methode zu entwickeln, wurden eine Reihe von verschiedenen Techniken versucht. Einzelne Larven Dissektion bis zu 1,5 Stunden dauern würde wurden, und durchschnittlich ~ 8000 Zellen mit dieser Methode18, von denen meisten nicht bis zum Jahresende Sammlung noch am Leben waren. Als nächstes ...

Watch this Scientific Journal Video about Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis at JoVE.com

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

Diese Methode zeigt, wie Erreger Invasion in Insektenzellen mit dreidimensionalen (3D) Modellen zu visualisieren. Hemocytes von Drosophila Larven infiziert wurden mit viralen oder bakteriellen Erregern, Ex Vivo oder in Vivo. Infizierte Hemocytes wurden dann fixiert und für die Bildgebung mit einem confocal Mikroskop und anschließende zelluläre 3D-Rekonstruktion befleckt.

Extraktion von Hemocytes aus Drosophila Melanogaster Larven für mikrobielle Infektionen und Analyse

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

Research

JoVE Journal

Immunology and Infection

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Ein Protokoll für die Erfassung und Färbung Blutzellen aus dem Gelbfiebermücke Aedes aegypti</em

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

Forschung

Immunologie und Infektion

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A Simple Chelex Protokoll zur DNA-Extraktion aus<em&gt; Anopheles</em&gt; Spp.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Isolation und Genomanalyse Einzel Virionen mit &#39;Single Virus Genomics&#39;

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Gesamtproteinextraktion und 2-D Gelelektrophorese-Methoden für<em&gt; Burkholderia</em&gt; Arten

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Systemische bakterielle Infektion und Immunabwehr Phänotypen in<em&gt; Drosophila melanogaster</em

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of ...

Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis

Paraffin einbetten und dünn schneiden von mikrobiellen Kolonie Biofilmen für die mikroskopische Analyse

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and ...

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

Kombination von Analyse von DNA in eine grobe Virion-Extraktion mit der Analyse der RNA aus den infizierten Blättern, entdecken neue Virus-Genom

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...

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

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

Gründung der Virusinfektion und Analyse von Host-Virus-Wechselwirkung in Drosophila Melanogaster

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 ...

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Die Galleria Mellonella Waxworm Infektionsmodell für verbreitet Candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Bewertung der Wirt-Pathogen-Antworten und Wirksamkeit des Impfstoffes bei Mäusen

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...