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

MCherry 表現コクシエラ ラベンダーの株を提供するため博士ロバート ・ Heinzen に感謝しております。博士ルイス ・ テイシェイラにありがとうはえの在庫を提供する無脊椎動物虹色ウイルス 6 およびブルーミントン ストック センターを提供します。このプロジェクトは、NIH グラント R00 AI106963 (A.G.G.) に、ワシントン州立大学によって一部で賄われていた。

1 感染前のヴィヴォ。
<ol><li>媒体および装置<ol>
<li>生殖不能の条件の下で新鮮なショウジョウバエ血球分離培地 (DHIM) 75% シュナイダーのショウジョウバエを含む準備中] 25% で胎仔ウシ血清 (FBS)、フィルター殺菌それ。</li>
		<li>顕微鏡下で 10 cm × 10 cm パラフィン フィルムの 2 〜 3 個をレイヤーします。</li>
		<li>ガラス毛細管を準備します。キャピラリーの引き手のヒータ?...

<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. 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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. 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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. 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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. 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宿主細胞が感染になる方法を理解、特に、以前テストされていない病原体および細胞型の組み合わせ4実験細胞、病原体の局在を明らかにすることが重要です。感染細胞反応カスケードを勉強して生産的な病原体の侵入を示すことができます、イメージングと細胞応答データの組み合わせ病原体の侵入、感染を示すことが欠かせません。一方、宿主細胞に病原体の侵入の 2D ?...

キイロショウジョウバエは免疫1の研究の確立されたモデル生物です。生得の免疫反応の間に血液細胞は病原体の挑戦への応答に重要な役割を果たします。血液細胞は、ウイルス、真菌や細菌感染2,3貪食作用を通じて病原体との闘いにおける重要な機能を持っていることと同様、寄生虫をカプセル化するために重要です。
最高病原微生物感染ホストの生得の免疫反応を理解し、病原体が伝染の間に宿主細胞に侵入する方法を視覚化することが重要です。この可視化は、侵入のメカニズムの理解に貢献しています。細胞応答し病原体の細胞内局在性の詳細と、これらのデータは感染症と微生物の相互作用、細胞細胞器官に対する宿主の反応についての手がかりを提供できます。したがって、顕微鏡によるイメージング後 3次元モデルの再構成は、宿主細胞に病原体の正確な位置を決定することができます。本研究ではコクシエラ中 (c.burnetii)、 Q 熱、プライマリショウジョウバエ血球に人間や動物の健康に深刻な脅威をもたらす人畜伝染病疾患の病原の侵略を可視化。最近、ショウジョウバエが 2 の 9 マイル相 II (NMII) クローンのc.burnetiiの 4 種、この株はショウジョウバエ4、複製することが、そのを示すバイオ セーフティ レベルに敏感であることを示したショウジョウバエ c.burnetii病因を研究するモデル生物として使用できます。
以前の研究では、ホストの生得の免疫反応を調べる血球を使用しています。血球は、形態学的観察5,6,7, 形態計測学的解析2,8、貪食分析2,3, qRT PCR2に使用されています。,9, 免疫沈降10,11、免疫蛍光分析10,12, 免疫染色13, イムノブロット3,10、 11および免疫組織化学の9,14。ショウジョウバエS2 細胞が様々 な体外実験の利用も、不死化および既存ウイルス感染の可能性は、動作15,16を変更します。S2 細胞などの不死化細胞ラインではなく一次電池の使用は、自然免疫の研究システムで全体の生物の代表的な。また、血液細胞の生体内での抽出の前に感染症は、他のホスト蛋白質および組織前のヴィヴォ感染前に血液細胞の抽出上の優位性と対話するセルをできます。血液細胞の生きている8,17,18,19を保つために時間の短い期間での血球の十分な数を取得するさまざまな方法の番号を利用されています。
本研究ではショウジョウバエ3rd病原微生物感染c.burnetii、リステリア菌(リステリア菌) や無脊椎動物の虹色の幼虫から血球を抽出する手法を提案します。ウイルス 6 (IIV6)。体内と体外の両方の血球感染症法について述べる。生体内でおよび前のヴィヴォ-感染した血液細胞の共焦点顕微鏡による可視化し、, c.burnetii侵略の 3 D モデルを構築するために使用します。また前のヴィヴォの抽出のプロトコルを使用して-感染した血液細胞は遺伝子および蛋白質の表現のために使用されたアッセイ。具体的には、IIV6 とリステリア感染症の程度を確認する総 RNA やタンパク質が分離された qRT PCR または西部のしみの分析のための細胞から。一緒に取られて、プロトコル 3rd幼虫と体内または体外に主な血液細胞が感染している証拠から急速に血球数を収集するメソッドを提供します、適切なプラットフォームの微生物病原体感染症研究および適用の下流解析顕微鏡、トランスクリプトミクス、プロテオミクスなど。

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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

ショウジョウバエ、病原菌の感染中に血球は感染で免疫応答に重要な役割を果たします。したがって、このプロトコルの目標は、ハエ、すなわち血液細胞の特定の免疫のコンパートメントの病原体の侵入を可視化する手法を開発することです。最大 3 × 10、ここで提示法を用いた6ライブ血球は、 ex vivo感染症のため 30 分で 200ショウジョウバエ3rd齢幼虫から入手できます。また、血球は感染した体内感染後 24 時間を追った血球抽出による 3rd幼虫の注射を通してできます。これらの感染した一次電池は修正され、ステンド グラス、共焦点顕微鏡を用いたイメージングします。その後、3 D 表現は、病原体の侵入を決定的に表示する画像から生成されました。また、qRT PCR のための質の高い RNA は mRNA 次の病原体の検出のため得ることができる感染症、および十分な蛋白質、西部のしみの分析のためのこれらの細胞から抽出されます。病原体の侵入の明確な和解と細菌やウイルスの病原体の種類と血球抽出のための効率的な方法を使用して、ショウジョウバエから十分なライブの血球を取得する感染症の確認法を提案する一緒に取られて、ex vivoとin vivoの感染実験の幼虫。

収集ex vivo感染症、血球を 3 × 10 まで生きて6血球は 200ショウジョウバエ3rd幼虫から抽出されました。本手法を開発するには、さまざまなテクニックの数を行った。個々 の幼生解離 1.5 h までと 8000 ~ セルの平均を得たこの法18、その大半はコレクションの終わりまで生きてなかった。次に、我々 は、血液を含んだ体液を?...

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

このメソッドは、三次元 (3 D) モデルを昆虫細胞に病原体の侵入を視覚化する方法を示します。ショウジョウバエ幼虫から血液細胞は、細菌やウイルスの病原体、体外か体内に感染していた。感染した血球、固定され共焦点顕微鏡とそれに続く細胞三次元イメージングのステンド グラスします。

細菌感染や解析のためのキイロショウジョウバエ幼虫血球の抽出

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

黄熱病の蚊から血球を収集し、染色のためのプロトコルネッタイシマカ</em

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

免疫・感染学

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

からのDNA抽出のためのシンプルなChelexプロトコル<em&gt;ハマダラカ</em&gt;属。

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'

&#39;シングルウイルスゲノミクス&#39;を使用してシングルビリオンの単離とゲノム解析

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

全タンパク質の抽出および2次元ゲル電気泳動法のための<em&gt;バークホルデリア</em&gt;種

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

全身細菌感染や免疫防御表現型<em&gt;キイロショウジョウバエ</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

パラフィン包埋と顕微解析の微生物コロニー バイオ フィルムの薄い断面

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

新しいウイルスのゲノムを発見する感染葉からの RNA の解析とウイルス粒子粗抽出 DNA の結合解析

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

ウイルス感染とキイロショウジョウバエのホスト ウイルスの相互作用の解析

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

播種性カンジダ症のハチミツガWaxworm 感染症モデル

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

宿主-病原体応答とマウスでワクチンの有効性の評価

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