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

Vi er takknemlige for Dr. Robert Heinzen for å gi bestander av mCherry-uttrykke Coxiella burnetii. Vi takker Dr. Luis Teixeira for å gi virvelløse iriserende virus 6 og Bloomington lager Center for å gi fly aksjer. Prosjektet ble finansiert delvis NIH grant R00 AI106963 (til A.G.G.) og Washington State University.

1. ex vivo infeksjon
<ol><li>Medium og utstyr<ol>
<li>Under sterile forhold, forberede frisk Drosophila Hemocyte isolere Medium (DHIM) som inneholder 75% Schneider's Drosophila medium med 25% fosterets Bovine Serum (FBS) og filter steriliseres.</li>
		<li>Lag 2-3 stykker av 10 cm x 10 cm parafin film under en stereomicroscope.</li>
		<li>Forberede av glass kapillær. Angi kapillær avtrekker varmeren 55% av maksimal. Dra kapillarrør til en skarp spissen av ca 10 µm.</li>
		<li>Etterfylle av...

<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. 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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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For bedre å forstå hvordan verten cellene blir smittet, er det viktig å avklare lokaliseringen av patogen i cellene, spesielt når eksperimentere på tidligere testet patogen og celle type kombinasjoner4. Mens studere cellulær respons kaskade etter infeksjon kan angi produktiv patogen invasjon, er kombinasjonen av tenkelig og cellulær respons viktig å demonstrere patogen invasjon og infeksjon. Mens rapporter som viser 2D-bilder av patogen invasjonen i verten cellene pleier å indikere produk...

Drosophila melanogaster er en veletablert modell organisme for studier av medfødt immunitet1. Under medfødte immunforsvaret spiller hemocytes en viktig rolle i svaret til patogen utfordring. Hemocytes er avgjørende for innkapsle parasitter, samt har en viktig funksjon i å bekjempe patogen gjennom phagocytic aksjon under sopp, viral og bakteriell infeksjon2,3.
For å best forstå vertens medfødte immunforsvaret til sykdomsfremkallende mikrober infeksjon, er det viktig å visualisere hvordan patogen invaderer vertsceller under infeksjon. Denne effekten bidrar til en forståelse av mekanismen for invasjon. Detaljer av patogen intracellulær lokalisering og cellulær respons, kan disse dataene gi hint om vert respons på infeksjon og mobilnettet organeller som mikrobe samhandler. Dermed kan 3D modell gjenoppbygging etter avbildning av mikroskopi være nyttig å finne nøyaktige plasseringen av patogener i verten cellene. I denne studien visualisert vi invasjonen av Coxiella burnetii (C. burnetii), den utløsende agenten for Q feber, en zoonotiske sykdommer som utgjør en alvorlig trussel for både mennesker og dyr helse, i primære Drosophila hemocytes. Nylig ble det vist at Drosophila er utsatt til biosikkerhet nivå 2 ni mil fase II (NMII) klone 4 stammen av C. burnetii og at denne belastningen er stand til å gjenskape i Drosophila4, som angir at Drosophila kan brukes som en modell organisme for å studere C. burnetii patogenesen.
Tidligere studier har brukt hemocytes for å undersøke vertens medfødte immunforsvaret. Hemocytes har vært brukt i morfologiske observasjoner5,6,7, morphometric analyse2,8, fagocytose analyse2,3, qRT PCR2 , 9, immunoprecipitation10,11, immunofluorescent analyse10,12, immunostai-13, immunoblotting3,10, 11 og immunohistochemistry9,14. Selv om Drosophila S2 celler er også tilgjengelig for forskjellige i vitro eksperimenter, endre immortalization og potensielle eksisterende virusinfeksjon sin atferd15,16. Bruk av primære celler i motsetning til en udødeliggjort cellen linje, for eksempel S2 celler, tillater for studier av medfødte immunforsvar i et system mer representative for hele organismen. I tillegg gir infeksjon av hemocytes i vivo, før ekstraksjon, cellene å samhandle med andre vert proteiner og vev, en fordel over utvinning av hemocytes før ex vivo infeksjon. En rekke ulike metoder har blitt benyttet for å få et tilstrekkelig antall hemocytes i løpet kort tid å holde hemocytes i live8,17,18,19.
I denne studien presenterer vi en metode for å trekke ut hemocytes fra Drosophila 3rd skikkelsen Larvene sykdomsfremkallende mikrober infisert med C. burnetii, Listeria monocytogenes (Listeria) eller virvelløse dyr iriserende virus 6 (IIV6). Vi beskriver metodene for både i vivo og ex vivo hemocyte infeksjoner. I vivo- og ex vivo-infiserte hemocytes var visualisert med AC confocal mikroskopi og brukes til å lage 3D-modeller av C. burnetii invasjon. I tillegg ved hjelp av utvinning, ex vivo-infiserte hemocytes ble brukt til genet og protein uttrykk analyser. Spesielt for å undersøke omfanget av infeksjon med IIV6 og Listeria, ble totalt RNA eller protein isolert fra celler for qRT PCR eller Western blot analyse. Samlet protokollen inneholder metoder for å raskt samle høyt antall hemocytes fra 3rd skikkelsen larver og bevis at primære hemocytes, infisert i vivo eller ex vivo, er en passende plattform for mikrobiell patogen infeksjon studier og gjeldende nedstrøms analyser som mikroskopi, transcriptomics og Proteomikk.

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	<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

Under patogene infeksjon av Drosophila melanogaster, spiller hemocytes en viktig rolle i immunforsvaret gjennom infeksjonen. Dermed er målet med denne protokollen å utvikle en metode for å visualisere den patogen invasjonen i en spesifikk immun kupé av fluer, nemlig hemocytes. Ved hjelp av metoden som presenteres her, opptil 3 × 10 kan6 live hemocytes fås fra 200 Drosophila 3rd skikkelsen larver i 30 min ex vivo infisert. Alternativt kan hemocytes være infisert i vivo gjennom injeksjon av 3rd skikkelsen Larvene etterfulgt av hemocyte utvinning til 24 timer etter infeksjon. Disse infiserte primære celler ble løst, beiset og fotografert bruker AC confocal mikroskopi. Deretter ble 3D representasjoner generert fra bilder å vise definitivt patogen invasjon. I tillegg høykvalitets RNA for qRT PCR kan oppnås for påvisning av patogen mRNA følgende infeksjon og tilstrekkelig protein kan trekkes fra disse cellene for Western blot analyse. Sammen presenterer vi en metode for bestemt avstemming av patogen invasjonen og bekreftelse av infeksjon med bakteriell og viral patogen typer og effektiv metode for hemocyte utvinning få nok live hemocytes fra Drosophila Larvene for ex vivo og i vivo infeksjon eksperimenter.

Å samle lever hemocytes for ex vivo infeksjon, opp til 3 × 10 ble6 hemocytes Hentet fra 200 Drosophila 3rd skikkelsen larver. For å utvikle vår metode, er en rekke ulike teknikker forsøkt. Individuelle larver disseksjon ville ta opptil 1,5 t, og gjennomsnittlig ~ 8000 celler ble hentet ved hjelp av denne metoden18, de fleste som ikke var i live på slutten av samlingen. Neste, vi prøvde å trekke ut hemolymph, som finne...

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

Denne metoden viser hvordan du visualisere patogen invasjonen i insekt celler med tredimensjonale (3D) modeller. Hemocytes fra Drosophila Larvene var infisert med virus eller bakterielle patogener, ex vivo eller i vivo. Infiserte hemocytes ble deretter fast og farget for bildebehandling med AC confocal mikroskop og påfølgende 3D cellular rekonstruksjon.

Utvinning av Hemocytes fra Drosophila melanogaster larver for mikrobielle infeksjoner og analyse

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

This technique can help answer key questions in the cellular infection and microbiology field, such as how hemocytes contribute to the overall immune response in fruit flies. The main advantage of this technique is that it can be performed in the laboratory, using previously infected larvae. Alternatively, hemocytes from uninfected larvae can be used for ex vivo infection.Visual demonstration of this method is critical because the imaging steps are difficult to learn because of the complexities of the confocal microscope and the options for 3D model reconstruction. To begin the protocol, layer two to three pieces of ten centimeter by ten centimeter paraffin film under a stereo microscope. Next, prepare the glass capillary by setting the capillary polar heater to 55%of maximum.Pole the capillary tube to a sharp point of approximately ten micrometers. Backfill the capillary with mineral oil. Assemble the filled capillary tube onto the nanoinjector.And open the fused capillary tube tip by breaking off the tip with forceps. Eject as much oil as possible from the capillary tube tip, then fill the tube with drosophila hemocyte isolating medium, or DHIM. For easy visualization of the border of hemolymphan oil, include an air bubble between the oil and DHIM.Using forceps, gently pick ten third instar drosophila larvae from the inside wall of food vials, and place them into a 100 micrometer strainer. Pour five milliliters of sterile water onto the larvae, and shake the strainer for five seconds. Place the strainer onto a task wipe to remove the excess water.Transfer the larvae into a 1.5 milliliter microcentrifuge tube, anesthetize them with CO2 gas for five seconds. Then, place the larvae onto paraffin film under the stereo microscope, with the dorsal side facing up. Place the glass capillary lightly onto the larval posterior body to hold it in place, and disrupt posterior cuticle open using fine-pointed forceps.Allow the hemolymph to flow onto the paraffin film. Make a pool of hemolymph, including hemocytes, from 20 to 50 larvae at a time, on the paraffin film. Use the glass capillary on the nanoinjector to take up the pooled hemolymph.Eject the hemolymph into a 1.5 milliliter microcentrifuge tube containing 500 microliters of DHIM. Place yeast paste on a grape juice agar plate. Make a fine cut in the agar where the larvae can migrate to avoid drying.Assemble a 0.001 millimeter pointed Tungsten needle with holding forceps using paraffin film. Pipette 50 microliters of high titer, mCherry-expressing C.Burnetii onto the paraffin film under the stereo microscope, and place the larvae into the pool of bacteria. Prick the larvae with a Tungsten needle.Transfer the larvae onto an agar plate. Transfer the remaining pathogen medium onto the agar plate, and seal the plate with paraffin film. Keep the larvae on the plate in moist air until the desired time post-infection.Leave the C.Burnetii-infected larvae on the plate for 24 hours. Place a 12 millimeter round cover glass in a well of a 24-well plate. Pipette 500 microliters of DHIM into the well.Extract and take up hemolymph from previously-infected larvae, then eject the hemolymph into the well. Centrifuge the plate at 1000 G for five minutes. Configure the confocal microscope for three-color imaging of DAPI, EGFP, and mCherry.Place the sample on the microscope and focus on the sample using a 63x 1.4 numerical aperture objective. Locate desired hemocytes in the field of view for imaging. Then, adjust the laser power and detector gains to achieve the appropriate exposure of the sample.Check multiple Z planes to ensure the exposure level is appropriate for the entire sample thickness. Next, find the top and bottom position on the Z axis of a whole hemocyte. Set these positions as the start and end positions for Z sectioning.Use scanning zoom to image the area containing the hemocyte. Collect the image series at the appropriate resolution, such as 1024 by 1024 pixels in the X Y plane, and 0.3 micrometer spacing in the Z dimension. Import the Z sectioned image series file into the software associated with the confocal microscope for 3D model reconstruction.Select a cell-showing co-localization of nuclei stained with DAPI and mCherry-expressing C.Burnetii in an EGFP-expressing hemocyte. Crop the image series to contain only the single cell. Select the 3D viewer option, which reconstructs the 3D model using the software's pre-packaged algorithm.Choose the desired imaging type for 3D representation among blend, surface, and mixed options. In this method, hemocytes, nuclei, and C.Burnetii are shown using surface models. Observe the 3D reconstructed cell from various viewing positions by holding the mouse button and dragging the cursor around the screen.Adjust the cell orientation and position of the modeled light source to optimize the image. Finally, take cross sections through the model using clipping and sectioning commands to visualize the interior contents of the hemocyte. Visualization of the extracted living hemocytes expressing EGFP under control of the hemolectin driver from multiple images show that most DAPI-positive cells are also EGFP-positive.Cross sections of Coxiella Burnetii-infected hemocytes confirm the presence of mCherry-expressing bacteria in the interior of the cell. Images were reconstructed into 3D models and we observed that in vivo infected hemocytes exhibited greater cytoplasmic extensions and were flatter in nature. While ex vivo infected hemocytes were more spherical.Cross sections of either model confirm the presence of mCherry-expressing Coxiella in the interior of the cell. Extracted hemocytes were infected with IIV6 and applied to qRT PCR analysis, which showed significantly higher levels of the IIV6-193R transcript between mock and IIV6-infected cells. While attempting this procedure, it's important to remember to have all media prepared ahead of time and equilibrated to room temperature so that cells are not without the proper nutrients for too long.Following this procedure, other methods, like single-cell RNA seek, can be performed to answer additional questions, like the overall host response to infection. After its development, this technique helps researchers in immunology and microbiology explore pathogen invasion and host responses in fruit flies, or other animals with open circulatory systems.

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

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

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

En enkel Chelex Protocol for DNA-ekstraksjon fra<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'

Isolasjon og Genome Analysis of single Virioner bruker &#39;enkelt 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

Totalt Protein Extraction og 2-D-gel-elektroforese Metoder<em&gt; Burkholderia</em&gt; Arter

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

Systemisk bakteriell infeksjon og immunforsvar fenotyper i<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

Parafin innebygging og tynn snitting av mikrobielle kolonien biofilm for mikroskopisk 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

Kombinere analyse av DNA i en grov Virion utvinning med analyse av RNA infiserte blader å oppdage nye Virus genomer

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

Etablering av Viral infeksjon og analyse av Host-Virus samhandling i 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

Galleria mellonella Waxworm infeksjon modell for spres 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

Evaluering av Host-patogen svar og vaksinen i mus

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