This research was possible through funding from the University of Neuch&#226;tel. We would like to thank all the students that helped in improving this technique, namely our colleague Kevin Thievent. We would also like to thank the members of the Thomas Lab for making their laboratory available. We would like to thank Janet Teeple for her help with mosquito rearing. We would also like to thanks Loyal Hall in the laboratory of Pr. Tom Baker for his help in the preparation of the micro capillary glass tubes.

注射や解剖1.生理食塩水<ol><li> pH値= 6.8で1.3のNaCl、0.5mMのKClと0.2 mMのCaCl 2をを得るために、蒸留水に塩化ナトリウム、塩化カリウム、および塩化カルシウムを添加することにより、生理食塩水を準備します。 </li><li>透明ビーズを着色するために生理食塩水の99ミリリットルに0.1％メチルグリーン溶液1mlを追加します。これは、0.001％メチルグリーン」注射液」です。 </li><li>その後、生理食塩水45mlの0.1％メチルグリーン溶液5mlを追加します。この0.01％メチルグリーン」解剖溶液」切開中のビーズの着色とその観察を容易にするために、メチルグリーンで10倍に濃縮します。 注：必要に応じてバッファを殺菌フィルター。 </li></ol> 2.キャピラリーの準備<ol><li>熱プルマイクロキャピラリーガラス管最小のビーズ（φ= 40μm）の42よりもわずかに大きい非常に微細な先端を得ることができます。 </li><li>オープンとAピンセットでチップを破壊したり、先端をオフにソーイングすることにより、各キャピラリをdjust。 </li></ol> 3.蚊飼育<ol><li> 26±1℃でリアのA.ハマダラカ 、70±5％の相対湿度、12：12時間の明：暗サイクル。成人女性を維持し、それらを10％糖液へのアクセスを与えます。 </li></ol>キャピラリー4.ビーズ選択<ol><li> 5センチメートルペトリ皿に負に帯電したビーズ（直径40〜120ミクロン）の0.009グラムを注ぎ、事前に注射液の30分の5ミリリットルを追加します。 注：目標は、注射のためのビーズ選択を容易にするために、染料の色にビーズをできるようにすることです。彼らは無菌ジャーに来ていない場合は、ビーズをオートクレーブ。 </li><li>キャピラリーピペット電球に搭載されたキャピラリーを用いて、視覚的に双眼顕微鏡下での生理食塩水0.1μlの最小のビーズを選択します。 注：同時に複数のビーズを接種すると、0.1マイクロリットルの総容積から3ビーズを選択します。</li></ol> 5.モスキート処理および接種<ol><li>昆虫吸引器を使用して、50ミリリットルチューブに各蚊を置きます。 </li><li>砕いた氷（2-5分）にチューブを配置することによって、簡単に各蚊を冷やします。 </li><li>双眼顕微鏡下でその右側に蚊を置きます。 </li><li>実体顕微鏡下で、しっかりと胸腔のキューティクルの左側を貫通した後、液体やビーズを注入することにより、キャピラリーで蚊を接種します。 注：可能な限り蚊に同様に垂直にキャピラリーを保持することにより、飛翔筋を傷つけないように注意してください。 </li><li>毛細血管を削除します。 </li><li>フェザー級昆虫学の鉗子を使用して、蚊帳で覆われた180ミリリットルのプラスチックカップに個別に女性を配置。彼らに10％の糖溶液へのアクセスを与えます。 注：蚊は、糖液滴にはまり込むことができるように注意、糖液で必要とされます。 </li></ol> 6.モスキート解剖とビーズの写真<ol><li>蚊が接種後24時間生きているかどうかを確認してください。もしそうであれば、その状態を確認し、飛ぶことができますだけで蚊を保ちます。 </li><li> -20℃で、これらの蚊をフリーズします。 注：必要に応じて彼らは、解剖の前に数ヶ月に数日間維持することができます。 </li><li> 32Xの倍率で双眼顕微鏡下でピンセットを使って蚊羽を削除します。透明テープでスライドガラス上に翼を固定してください。各個々の蚊のために使用されるコードを書き留めます。 </li><li>スキャナでガラススライドを入れて、1200 dpiでそれらをスキャンします。 注：コードは、個々の蚊のために読み取り可能であることを確認してください。 </li><li>ガラス顕微鏡スライド上にと解剖溶液中で、頭部と腹部から胸部を分離するために鉗子を使用しています。 </li><li>ビーズを取得するために鉗子で胸部を開きます。 注：腹部に移動しないビーズは、互いに接触しておらず、それらのほとんどがTで自由に浮きます彼は血リンパ。非メラニンビーズを見つけるのは厳しいものになる可能性があるため、ピンセットでビーズを壊さないように注意してください。色を得るためにビーズ1〜5分間待ってください。 </li><li>写真を撮る前に、ガラス顕微鏡スライド上の解剖液の液滴中にビーズを転送します。 注：ビーズを洗浄する必要はありません。 </li><li>カメラを備えた顕微鏡を使用して、標準の照明設定と400Xの倍率で各ビーズのデジタル画像を撮ります。 注：彼らの比較を可能にするために、異なる画像間の照明を変更しないでください。 </li></ol> 7.画像解析：ビーズメラニン<ol><li>画像解析ソフトウェアを開きます。 「ファイル」をクリックして、「開く」をビーズ画像を見つけること。 </li><li>クリックして「分析」。クリックして「設定の測定値。 &quot; 「セット測定」ウィンドウで「グレイ平均値」ボックスにチェックを入れ。 </li><li>サークル選択ツールアイコンをクリックします。ビーズ周囲を選択します。に残りますビーズを左右し、その周りに明るいハローを避けます。 </li><li> 「分析」メニューをクリックします。クリックして「尺度。」ビーズの断面積の平均グレー値とサイズを取得します。 <ol><li> 「 - 灰色の平均値256。」：各ビーズのメラニン値を取得するには、電卓や計算機のソフトウェアを使用して、次の操作を行いますメラニンの尺度は0が完全に白画像を表すと、0と256の間の値です。 </li></ol></li></ol>

<ol>
	<li>Dong, Y., Aguilar, R., Xi, Z., Warr, E., Mongin, E., Dimopoulos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anopheles+gambiae+immune+responses+to+human+and+rodent+Plasmodium+parasite+species.">Anopheles gambiae immune responses to human and rodent Plasmodium parasite species.</a> PLoS pathog. 2 (6), e52 (2006).</li><li>Sadd, B. M., Schmid-Hempel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PERSPECTIVE:+Principles+of+ecological+immunology.">PERSPECTIVE: Principles of ecological immunology.</a> Evolutionary Appl. 2 (1), 113-121 (2008).</li><li>Crompton, P. D., Moebius, 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=Malaria+Immunity+in+Man+and+Mosquito:+Insights+into+Unsolved+Mysteries+of+a+Deadly+Infectious+Disease*.">Malaria Immunity in Man and Mosquito: Insights into Unsolved Mysteries of a Deadly Infectious Disease*.</a> Annu Rev Immuno. 32 (1), 157-187 (2014).</li><li>Schmid-Hempel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EVOLUTIONARY+ECOLOGY+OF+INSECT+IMMUNE+DEFENSES.">EVOLUTIONARY ECOLOGY OF INSECT IMMUNE DEFENSES.</a> Annu Rev Entomol. 50 (1), 529-551 (2005).</li><li>Hillyer, J. F., Schmidt, S. L., Christensen, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+phagocytosis+and+melanization+of+bacteria+and+Plasmodium+sporozoites+by+hemocytes+of+the+mosquito+Aedes+aegypti.">Rapid phagocytosis and melanization of bacteria and Plasmodium sporozoites by hemocytes of the mosquito Aedes aegypti.</a> J parasito. 89 (1), 62-69 (2003).</li><li>Carissimo, G., Pondeville, 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=Antiviral+immunity+of+Anopheles+gambiae+is+highly+compartmentalized,+with+distinct+roles+for+RNA+interference+and+gut+microbiota.">Antiviral immunity of Anopheles gambiae is highly compartmentalized, with distinct roles for RNA interference and gut microbiota.</a> PNAS. 112 (2), E176-E185 (2015).</li><li>Christensen, B. M., Li, J., Chen, C. -. C., Nappi, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanization+immune+responses+in+mosquito+vectors.">Melanization immune responses in mosquito vectors.</a> Trends parasito. 21 (4), 192-199 (2005).</li><li>Collins, F., Sakai, 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=Genetic+selection+of+a+Plasmodium-refractory+strain+of+the+malaria+vector+Anopheles+gambiae.">Genetic selection of a Plasmodium-refractory strain of the malaria vector Anopheles gambiae.</a> Science. 234 (4776), 607-610 (1986).</li><li>Warr, E., Lambrechts, L., Koella, J. C., Bourgouin, C., Dimopoulos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anopheles+gambiae+immune+responses+to+Sephadex+beads:+Involvement+of+anti-Plasmodium+factors+in+regulating+melanization.">Anopheles gambiae immune responses to Sephadex beads: Involvement of anti-Plasmodium factors in regulating melanization.</a> Insect Biochem Molec. 36 (10), 769-778 (2006).</li><li>Fuchs, S., Behrends, V., Bundy, J. G., Crisanti, A., Nolan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenylalanine+metabolism+regulates+reproduction+and+parasite+melanization+in+the+malaria+mosquito.">Phenylalanine metabolism regulates reproduction and parasite melanization in the malaria mosquito.</a> PloS one. 9 (1), e84865 (2014).</li><li>Cerenius, L., S&#246;derh&#228;ll, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prophenoloxidase-activating+system+in+invertebrates.">The prophenoloxidase-activating system in invertebrates.</a> Immuno Rev. 198, 116-126 (2004).</li><li>Cerenius, L., Lee, B. L., S&#246;derh&#228;ll, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+proPO-system:+pros+and+cons+for+its+role+in+invertebrate+immunity.">The proPO-system: pros and cons for its role in invertebrate immunity.</a> Trend Immuno. 29 (6), 263-271 (2008).</li><li>Moret, Y., Schmid-Hempel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Survival for immunity: the price of immune system activation for bumblebee workers. , 1166-1168 (2000).</li><li>Suwanchaichinda, C., Paskewitz, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Larval+Nutrition,+Adult+Body+Size,+and+Adult+Temperature+on+the+Ability+of+Anopheles+gambiae(Diptera:+Culicidae)+to+Melanize+Sephadex+Beads.">Effects of Larval Nutrition, Adult Body Size, and Adult Temperature on the Ability of Anopheles gambiae(Diptera: Culicidae) to Melanize Sephadex Beads.</a> J Med Entomol. 35 (2), 157-161 (1998).</li><li>Chun, J., Riehle, M., Paskewitz, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Mosquito+Age+and+Reproductive+Status+on+Melanization+of+Sephadex+Beads+in+Plasmodium-Refractory+and+-Susceptible+Strains+of+Anopheles+gambiae.">Effect of Mosquito Age and Reproductive Status on Melanization of Sephadex Beads in Plasmodium-Refractory and -Susceptible Strains of Anopheles gambiae.</a> J Invertebr Pathol. 66 (1), 11-17 (1995).</li><li>Schwartz, A., Koella, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanization+of+Plasmodium+falciparum+and+C-25+Sephadex+Beads+by+Field-Caught+Anopheles+gambiae+(Diptera:+Culicidae)+from+Southern+Tanzania.">Melanization of Plasmodium falciparum and C-25 Sephadex Beads by Field-Caught Anopheles gambiae (Diptera: Culicidae) from Southern Tanzania.</a> J Med Entomol. 39 (1), 84-88 (2002).</li><li>Zahedi, M., Denham, D. A., Ham, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+and+melanization+responses+of+Armigeres+subalbatus+against+inoculated+Sephadex+beads.">Encapsulation and melanization responses of Armigeres subalbatus against inoculated Sephadex beads.</a> J Invertebr Pathol. 59 (3), 258-263 (1992).</li><li>Laughton, A. M., Garcia, J. R., Altincicek, B., Strand, M. R., Gerardo, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+immune+responses+in+the+pea+aphid,+Acyrthosiphon+pisum.">Characterisation of immune responses in the pea aphid, Acyrthosiphon pisum.</a> J insect physiol. 57 (6), 830-839 (2011).</li><li>Barreaux, A. M. G., Barreaux, P., Koella, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overloading+the+immunity+of+the+mosquito+Anopheles+gambiae+with+multiple+immune+challenges.">Overloading the immunity of the mosquito Anopheles gambiae with multiple immune challenges.</a> Parasite Vector. 9 (1), 210 (2016).</li><li>Lazzaro, B. P., Rolff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Danger,+Microbes,+and+Homeostasis.">Danger, Microbes, and Homeostasis.</a> Science. 332 (6025), 43-44 (2011).</li><li>Arrighi, R. B. G., Faye, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+falciparum+GPI+toxin:+a+common+foe+for+man+and+mosquito.">Plasmodium falciparum GPI toxin: a common foe for man and mosquito.</a> Acta trop. 114 (3), 162-165 (2010).</li><li>Michel, K., Kafatos, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquito+immunity+against+Plasmodium.">Mosquito immunity against Plasmodium.</a> Insect Biochem Molec. 35 (7), 677-689 (2005).</li><li>Osta, M. A., Christophides, G. K., Vlachou, D., Kafatos, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immunity+in+the+malaria+vector+Anopheles+gambiae:+comparative+and+functional+genomics.">Innate immunity in the malaria vector Anopheles gambiae: comparative and functional genomics.</a> J Exp Biol. 207 (15), 2551-2563 (2004).</li><li>Christophides, G. K., Vlachou, D., Kafatos, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+and+functional+genomics+of+the+innate+immune+system+in+the+malaria+vector+Anopheles+gambiae.">Comparative and functional genomics of the innate immune system in the malaria vector Anopheles gambiae.</a> Immunol Rev. 198 (1), 127-148 (2004).</li><li>Blair, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquito+RNAi+is+the+major+innate+immune+pathway+controlling+arbovirus+infection+and+transmission.">Mosquito RNAi is the major innate immune pathway controlling arbovirus infection and transmission.</a> Future microbiol. 6 (3), 265-277 (2011).</li><li>Fragkoudis, R., Attarzadeh-Yazdi, G., Nash, A. A., Fazakerley, J. K., Kohl, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+dissecting+mosquito+innate+immune+responses+to+arbovirus+infection.">Advances in dissecting mosquito innate immune responses to arbovirus infection.</a> J Gen Virol. , (2009).</li><li>Schwartz, A., Koella, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cost+of+immunity+in+the+yellow+fever+mosquito,+Aedes+aegypti+depends+on+immune+activation.">The cost of immunity in the yellow fever mosquito, Aedes aegypti depends on immune activation.</a> J evol biol. 17 (4), 834-840 (2004).</li><li>Lambrechts, L., Vulule, J. M., Koella, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+correlation+between+melanization+and+antibaterial+immune+responses+in+a+natural+population+of+the+malaria+vector+Anopheles+gambiae.">Genetic correlation between melanization and antibaterial immune responses in a natural population of the malaria vector Anopheles gambiae.</a> Evolution. 58 (10), 2377 (2004).</li><li>Boete, C., Paul, R. E. L., Koella, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+and+indirect+immunosuppression+by+a+malaria+parasite+in+its+mosquito+vector.">Direct and indirect immunosuppression by a malaria parasite in its mosquito vector.</a> P Roy Soc B-Biol Sci. 271 (1548), 1611-1615 (2004).</li><li>Sacks, D., Sher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evasion+of+innate+immunity+by+parasitic+protozoa.">Evasion of innate immunity by parasitic protozoa.</a> Nat immunol. 3 (11), 1041-1047 (2002).</li><li>Zambrano-Villa, S., Rosales-Borjas, D., Carrero, J. C., Ortiz-Ortiz, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+protozoan+parasites+evade+the+immune+response.">How protozoan parasites evade the immune response.</a> Trend Parasito. 18 (6), 272-278 (2002).</li><li>Damian, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parasite+immune+evasion+and+exploitation:+reflections+and+projections.">Parasite immune evasion and exploitation: reflections and projections.</a> Parasitology. 115, S169-S175 (1997).</li><li>Schmid-Hempel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parasite+immune+evasion:+a+momentous+molecular+war.">Parasite immune evasion: a momentous molecular war.</a> Trend ecol evol. 23 (6), 318-326 (2008).</li><li>Schmid-Hempel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+defence,+parasite+evasion+strategies+and+their+relevance+for+&amp;#34;macroscopic+phenomena&amp;#34;+such+as+virulence.">Immune defence, parasite evasion strategies and their relevance for &amp;#34;macroscopic phenomena&amp;#34; such as virulence.</a> P Roy Soc B-Biol Sci. 364 (1513), 85-98 (2009).</li><li>Stearns, S. C., Koella, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+phenotypic+plasticity+in+life+history+traits-+predictions+of+reaction+norms+for+age+and+size+at+maturity.">The evolution of phenotypic plasticity in life history traits- predictions of reaction norms for age and size at maturity.</a> Evolution. 40 (5), 893-913 (1986).</li><li>Stearns, 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=Life-history+tactics:+a+review+of+the+ideas.">Life-history tactics: a review of the ideas.</a> Q rev biol. 51 (1), 3-47 (1976).</li><li>Valtonen, T. M., Kleino, A., Ramet, M., Rantala, 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=Starvation+Reveals+Maintenance+Cost+of+Humoral+Immunity.">Starvation Reveals Maintenance Cost of Humoral Immunity.</a> Evol Biol. 37 (1), 49-57 (2010).</li><li>Sheldon, B. C., Verhulst, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological+immunology:+costly+parasite+defences+and+trade-offs+in+evolutionary+ecology.">Ecological immunology: costly parasite defences and trade-offs in evolutionary ecology.</a> Trend Ecol Evo. 11 (8), 317-321 (1996).</li><li>Graham, A. L., Allen, J. E., Read, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+causes+and+consequences+of+immunopathology.">Evolutionary causes and consequences of immunopathology.</a> Annu Rev Ecol Evol S. 36, 373-397 (2005).</li><li>Best, A., Long, G., White, A., Boots, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+implications+of+immunopathology+for+parasite+evolution.">The implications of immunopathology for parasite evolution.</a> P Roy Soc B-Biol Sci. 279 (1741), 3234-3240 (2012).</li><li>Cator, L. J., George, 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=34;Manipulation&amp;#34;+without+the+parasite:+altered+feeding+behaviour+of+mosquitoes+is+not+dependent+on+infection+with+malaria+parasites.">34;Manipulation&amp;#34; without the parasite: altered feeding behaviour of mosquitoes is not dependent on infection with malaria parasites.</a> P Roy Soc B-Biol Sci. 280 (1763), 20130711 (2013).</li><li>Voordouw, M. J., Lambrechts, L., Koella, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+maternal+effects+after+stimulation+of+the+melanization+response+in+the+yellow+fever+mosquito+Aedes+aegypti.">No maternal effects after stimulation of the melanization response in the yellow fever mosquito Aedes aegypti.</a> Oikos. 117 (8), 1269-1279 (2008).</li><li>Paskewitz, S., Riehle, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+Plasmodium+refractory+and+susceptible+strains+of+Anopheles+gambiae+to+inoculated+Sephadex+beads.">Response of Plasmodium refractory and susceptible strains of Anopheles gambiae to inoculated Sephadex beads.</a> Dev comp immunol. 18 (5), 369-375 (1994).</li></ol>

The authors declare that they have no competing financial interests.

この注入法は、蚊におけるメラニンの応答を刺激し、検討することが有用です。例えば、ここでは、免疫刺激負荷の影響を検討しました。 この手順の重要なステップは、適切に蚊に接種することです。飛翔筋または蚊自体への過度な損傷が給紙から蚊を防ぐことができるか、解剖の前にそれを殺すことができます。第二の重要なステップは、それらを殺すことなく、それらをノックアウトするのに十分な長さ氷上で蚊を維持することです。それは氷との緊密な接触を可能にするような小さな容器は、プロセスを容易にし、スピードアップします。ピンセットは、そうでない場合は、ビーズを破損する可能性があり最後に、特別な注意が、解剖時に必要とされています。蚊の組織に埋め込まれたビーズは、解剖液で着色するより多くの時間がかかることがあります。 彼らはMと（のような中立的、積極的、または負に帯電したビーズやガラスビーズ）は、異なる結果につながるビーズの複数種類を使用しますAYすべて同じ程度27,43にメラニンされていません。実際に、中性に荷電したビーズに比べて、負に帯電したビーズは、蚊27との間のメラニン応答のより多くの変動につながります。ガラスビーズは、他の一方で、蚊に不活性であり、すべての27でメラニン応答を誘発しません。負に帯電したビーズは、このように好ましいかもしれないが、別のビーズとの間の比較はまた、適切であり得ます。しかし、ビーズの種類に応じて、異なる色素を注入し、解剖ソリューションのために必要な場合があります。ガラスビーズ27を使用する場合、例えば、クレシルバイオレット染料の代わりにメチルグリーンを使用すべきです。 この注入法は、蚊のために侵襲的です。破損や損傷が最小限に抑えられ、制御することができたとしても、代替のツールは、実験計画に応じて、より良いオプションかもしれません。同じ蚊のいくつかの免疫応答を刺激し、同時に測定することは困難です。作業免疫応答28との間の相関を見ると蚊の家族やグループでる必要です。また、ビーズは、昆虫における寄生虫の実際の存在に部分的にしか似ています。 まず、免疫応答が刺激され、その後、得られた表現型の応答、カプセル化は、測定されるが、何の間で起こることは知られていません。生理学的または分子ツールで10回の注射をカップリング- 12は、このメラニン応答の我々の理解を向上させることができます。 12 -メラニン応答の根底にあるメカニズムを研究するために、重要な酵素またはプロフェノールオキシダーゼカスケード中に生成分子の発現は、10を測定することができます。しかし、最終的なカプセル化の実際の結果を知らずに、アップまたはダウンレギュレーションこのような分子のもっぱら焦点を当て、メラニン形成プロセスの不正確な評価につながる可能性があります。そのため、勉強encapsulationと一緒に分子メカニズムは別々に使用するとき、彼らはそれぞれの有効な科学的手法であるという事実にもかかわらず、非常に有益であろう。 結論として、これは免疫力や免疫力の進化を研究する技術です。マラリア原虫に対する殺虫剤抵抗性および蚊の感受性との間のリンクの証拠を増加させる、例えば、存在します。この注入法は、フィールド試験で、ならびに、そのような状況で使用することができます。第二の作業方向を詳細に免疫経路を研究するために、遺伝子ノックダウンと免疫刺激を組み合わせることです。

そのキューティクルまたはそれらの中腸上皮4を通して破る3 -昆虫は寄生虫や病原体1から身を守るために免疫応答に依存しています。蚊では、これらの応答は、細菌5、ウイルス6、フィラリア線虫7、およびマラリア原虫1,8,9に対して効率的です。 12 -蚊では、キー免疫応答は、メラニン10で異物をカプセル化したものです。 12 -このカプセル化は、中腸内または血リンパ循環システム10に発生する可能性があります。このメラニン応答は、プロフェノールオキシダーゼカスケード10の結果である- 12、そしてそれは寄生虫の死に、またはそれらの食作用につながることができます。血球細胞の数は限られている大人の蚊では、メラニンは、マラリア原虫の寄生虫やフィラリア線虫7に対してのように、体液性応答です。いくつかの他の昆虫では、それは直接7メラニン化するために寄生虫の周りに集まる血球細胞です。また、メラニンは7治癒産卵とキューティクルの傷のようないくつかの他の生理学的プロセスのためにも不可欠です。 18 -免疫応答の刺激は、いくつかの農業および公衆衛生モデル系13の昆虫耐性を研究するためのツールとして使用されます。 16,19 -宿主-寄生虫相互作用14を研究するハマダラカ蚊、アフリカにおけるマラリアの主要なベクトルで使用されています。これらの技術は、それらのパターン認識受容体（PRR）2で寄生虫を検出するために、昆虫の能力に基づいています。蚊はまた、病原体関連分子パターン（PAMP）のようなそれらの生物学的に干渉する他の分子を検出する、またはコラーゲンおよび核酸の放出に起因する独自の損傷した細胞を検出することができます。蚊免疫細胞23 -このような血球などの検出20のために使用されています。主な免疫シグナル伝達経路は、IMD、トール、JAK / STAT 24、およびリボ核酸干渉（RNAi）25,26です。トールおよびIMD経路の両方は、メラニンの応答に影響を及ぼし、プロフェノールオキシダーゼカスケード10との対話- 12。 メラニンの応答を刺激するために使用される標準的なツールは、胸腔の血リンパ中に小さなビーズと蚊の接種です。メラニンのカプセル化の程度は、その後、蚊の切開を介してビーズを回収した後、19を測定することができます。ほとんどの研究では、唯一のビーズは蚊15,16,27毎に注入したが、より多くのビーズを注入すると、メラニン応答19の限界を研究するために可能です。これらのビーズは、蚊の生理機能の妨害を制限するために、注射液（生理血清）を使用して注入され、蚊15,16,27の乾燥。色素は、ビーズの選択を容易にするためにこの溶液に添加します。これは、ビーズ15,16,27を取得するために使用される解剖ソリューションについても同様です。 非病原性の刺激で虫を接種することの利点は、免疫応答に直接影響に焦点を当てることができることです。 31、または免疫回避31 - - 34による寄生病原28、免疫抑制29への複雑な効果はありません。また、このような寿命や産卵数などの他の生活史形質、上の刺激の結果は、また、研究することができます。したがって、進化生態学を研究する研究者は、このようなツール2,35,36が必要な場合があります。例えば、免疫挑戦マルハナバチは、飢餓の下に短縮寿命を持っています。免疫刺激と展開の同様の負の効果は、多くの場合、短いで、その結果、別の無脊椎動物のモデルで観察されていますえー寿命以下繁殖成功13,27,37。このような研究は、環境に2,4,38を変えて実施することができます。免疫力を刺激することも免疫病理39,40に直接焦点を当てたものに興味があります。 このプロトコルは、メラニン応答を刺激し、直接メラニンの量を測定するために蚊を有するビーズの接種に基づいています。これは、異なる実験設定でメラニン応答の定量的および定性的な研究を可能にします。このようなツールは、加熱死菌41に抗菌性応答などの他の免疫応答の刺激に拡張することができます。また、多くの生態系の設定で行うことができます。

<table border="0" cellpadding="0" cellspacing="0" width="718">
	<tbody>
		<tr height="85">
			<td height="85" style="height:85px;width:301px;">Microcapillary glass tubes GB120TF-10</td>
			<td style="width:177px;">science-products.com</td>
			<td style="width:84px;">GB120TF-10</td>
			<td style="width:156px;">http://www.science-products.com/Products/CatalogG/Glass/Glass.html</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:301px;">Microcaps Capillary pipette bulb</td>
			<td style="width:177px;">Drumond</td>
			<td style="width:84px;">1-000-9000</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:301px;">negatively charged Sephadex CM C-25 beads</td>
			<td style="width:177px;">Sigma-Aldrich, Steinheim, Germany</td>
			<td style="width:84px;">C25120 SIGMA</td>
			<td style="width:156px;">need few to start</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:301px;">Methyl green</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:84px;">323829 ALDRICH</td>
			<td style="width:156px;">need few to start</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:301px;">Software ImageJ</td>
			<td style="width:177px;">opensource</td>
			<td style="width:84px;"></td>
			<td style="width:156px;">Version 1.47f7 or later</td>
		</tr>
	</tbody>
</table>

inoculating anopheles gambiae mosquitoes with beads to induce and measure the melanization immune response

免疫応答の刺激は、有効性と免疫のメカニズムを検討する無脊椎動物研究における一般的なツールです。粒子は免疫系によって検出され、免疫エフェクターの産生を誘導するように、この刺激は、昆虫への非病原性粒子の注射に基づいています。私たちは、蚊ハマダラカにおけるメラニンの応答の刺激にここに焦点を当てます。メラニンの応答は、メラニンの暗い層で異物や寄生虫のカプセル化につながります。この応答を刺激するために、蚊は、微小ガラス管を用いて胸腔内のビーズを用いて接種します。そして、24時間後に、蚊がビーズを取得するために解剖されています。ビーズのメラニンの程度は画像分析ソフトウエアを用いて測定されます。ビーズは、病原性寄生虫の効果、または免疫応答を回避または抑制するための能力を持っていません。これらの注射は、measuする方法です免疫有効性及びそのような繁殖力や長寿などの他の生活史形質、上の免疫刺激の影響再。これは、直接宿主 - 寄生虫相互作用を研究するとまったく同じではありませんが、免疫力とその進化生態学を研究するための興味深いツールです。

<html>いくつかのビーズが少ない他よりもメラニン（ 図1）で覆われていたとして蚊は、同じようにビーズをメラニン化するすべてではないでした。実際、いくつかのビーズが（ 図1）他の人が完全に暗くしたのに対し、理由メラニンの不足の青のままでした。メラニン値（青およびunmelanizedビーズに相当）が0と100（暗いと高メラニンビーズに相当）（ 図2a及び2b）の間の値を線形補間することにより標準化しました。これらの値は、従って、免疫応答の強度は、ビーズのメラニンの程度を示し、。 私たちは、彼らのメラニン値を比較することにより、相互にビーズを比較することができます。メラニンの応答は、一般的ANOVAまたはガウスGLMのような古典的な統計分析を可能にする、ガウス分布に従います。 。 &quot;FO：キープtogether.within-ページ=&quot; 1 &quot;&gt;我々は同時に蚊に接種したビーズの数に対するメラニン応答の変化を研究の目標は、変動のより良い理解を得ることであったとの限界メラニン応答。ビーズのサイズは、任意の効果を持っていませんでした。 ビーズの数は、（Fのigure 2）増加した接種した場合、平均ビーズメラニン値は50に71から減少しました。免疫チャレンジが増加蚊におけるメラニン応答のいくつかの制限があります。また、メラニンの応答はそれぞれ1〜3個のビーズ、のために71から35に、各蚊に少なくとも-メラニンビーズの減少となりました。しかし、最も強くメラニンビードのメラニン値はほぼ一定にとどまりました。メラニンの変動は、ビーズの数とともに増加しました。メラニンの努力は、与えられた蚊内のすべてのビーズの間で均一ではありませんでした1ビーズのような他のものよりも優先されました。この注入法は、蚊に免疫応答の変動性を検討することが有用です。 <img alt="図1" src="/files/ftp_upload/55013/55013fig1.jpg" /> 図1：非メラニンの画像の例では、部分的にメラニン、および高度メラニンビーズ解剖後。左、中央、右には、それぞれ、非メラニンビーズ、部分的にメラニンビード、および高度メラニンのビーズです。 <img alt="図2" src="/files/ftp_upload/55013/55013fig2.jpg" /> 図2：接種したビーズの数の関数としてのビーズメラニン。 （a）は、各ポイントは（メラニン値が重くメラニンものに非メラニンビーズのための0から100までの範囲で）単一のビーズのメラニン化を示し、点の各列は、個々の蚊を表します。各mosquitoは、1つ、2つ、または3つのビーズを接種しました。固体ポイントは蚊あたり最高メラニン値を表し、交差した点は、中間メラニン値を表し、空のポイントが最も低いメラニン値を表します。実線はビーズ処置あたりの平均メラニンを表し、破線は、最も高いメラニンの平均値を示し、点線は最小メラニンの平均を示します。 （b）の平均とメラニン、最高のメラニンとビーズ接種の処置あたり最低メラニンの標準誤差。 ブラック（重くメラニンものに非メラニンビーズ0から100までの範囲のメラニン値を有する）ビーズの数の関数として平均ビーズメラニンを表します。赤い点が最も高いメラニンの平均を表します。青い三角形は、最低メラニンの平均を表します。バーは標準誤差です。参照19から変更されました。&lt;/ P&gt;

Watch this Scientific Journal Video about Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response at JoVE.com

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

Through inoculation with beads, the described technique enables the stimulation of the mosquito melanization response in the hemolymph circulating system. The amount of melanin covering the beads can be measured after dissection as a measure of the immune response.

接種<em&gt;ハマダラカ</emメラニン免疫応答を誘導し、測定するためにビーズと&gt;蚊

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is ...

inoculating-anopheles-gambiae-mosquitoes-with-beads-to-induce-measure

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

7.9K ビュー.  Université de Neuchâtel. Through inoculation with beads, the described technique enables the stimulation of the mosquito melanization response in the hemolymph circulating system. The amount of melanin covering the beads can be measured after dissection as a measure of the immune response.

ビデオ: Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

に感染したマウスにおける細菌負荷と免疫応答の測定リステリア菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

免疫・感染学

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

の寄生蜂の紹介<em&gt;ショウジョウバエ</em&gt;と抗寄生虫免疫応答

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

2光子顕微鏡を用いてマウスの胸腺の生体内イメージング

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

免疫応答やスリープ中の定量的測定<em&gt;ショウジョウバエ</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

ゼブラフィッシュにおける自然免疫の健康の指標として呼吸バースト応答の定量化

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

マウスマクロファージにおける先天性免疫応答を調査するために、RNA干渉を使用

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

マウスにおける宿主の免疫応答を評価する口腔咽頭気管内PAMP管理および気管支肺胞の活用

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

西ナイルウイルス変異株の感染に対するMyD88非介在細胞性免疫応答のインビトロ分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

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

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

リポ多糖投与マウス肝臓における免疫応答時に白血球の二光子励起生体イメージング

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

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

マウスのモデルを評価する生得の免疫反応黄色ブドウ球菌感染症

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

接種

要約

さらに動画を探す

この動画の章

に感染したマウスにおける細菌負荷と免疫応答の測定リステリア菌

の寄生蜂の紹介