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.

1.注射和解剖盐溶液<ol><li>通过加入氯化钠，氯化钾，及氯化钙2到蒸馏水在pH = 6.8，得到1.3毫摩尔NaCl，0.5mM的KCl和0.2mM的氯化钙制备的盐溶液。 </li><li>将1ml的0.1％甲基绿溶液添加到99毫升计盐溶液的着色透明珠。这是0.001％甲基绿"注射液"。 </li><li>然后，加入5毫升0.1％甲基绿溶液至45毫升生理盐水溶液。这个0.01％甲基绿"夹层溶液"是甲基绿10倍以上的浓缩，以促进珠粒在解剖的着色和它们的观察。 注意:如果需要过滤消毒的缓冲区。 </li></ol> 2.毛细准备<ol><li>热拉微细玻璃管以获得非常细的尖端比最小的小珠（φ= 40微米）42稍大。 </li><li>打开和通过打破用镊子尖或锯尖断djust每个毛细管。 </li></ol> 3.饲养蚊子<ol><li>后方冈比亚按蚊在26±1℃，70±5％相对湿度，和12:12小时的光:暗周期。保持成年女性，让他们获得了10％的糖溶液。 </li></ol> 4.用毛细管珠选择<ol><li>在5cm的培养皿中倒入0.009克的带负电荷的小球（40-120微米直径），并加入5毫升注射液30分钟提前。 注意:我们的目标是让染料着色珠，使珠选择用于注射的更容易。高压灭菌珠子，如果他们不进来的无菌罐子。 </li><li>使用安装在毛细吸管灯泡的毛细管，直观地选择一个双目显微镜下在0.1微升的盐溶液中的最小珠。 注意:当同时接种几个珠，0.1微升的总体积选择三个珠。</li></ol> 5.蚊子处理和接种<ol><li>使用昆虫吸引器，将每个蚊子在50毫升管中。 </li><li>通过将管在碎冰（2-5分钟）简要地冷却各蚊子。 </li><li>放置在其右侧蚊子的双目显微镜下。 </li><li>下一个立体显微镜，通过经由胸腔角质层的左侧牢牢刺穿，然后注入液和珠接种与毛细蚊子。 注意:小心不要握住毛细管作为垂直于在蚊子可能损坏飞行肌肉。 </li><li>取下毛细管。 </li><li>使用轻量级昆虫学镊子，将每个女性在覆盖着蚊帐180毫升的塑料杯中分别;让他们获得了10％的糖溶液。 注:警告是必需的与糖的溶液，作为蚊可能会卡在糖溶液滴。 </li></ol> 6.蚊子解剖和珠摄影<ol><li>检查蚊子是活在接种后24小时。如果是这样，检查他们的条件，只保留那些能飞的蚊子。 </li><li>冻结这些蚊子在-20℃。 注:他们可以解剖前几天保持到几个月，如果需要的话。 </li><li>在32X倍的双目显微镜下取出用镊子蚊子的翅膀。固定机翼上用透明胶带载玻片上。写下用于每个蚊子的代码。 </li><li>把玻璃载片在一台扫描仪，并在1200 dpi的扫描。 注意:检查代码是否可读每一个人蚊子。 </li><li>在玻璃显微镜载片，并在夹层溶液，使用镊子胸廓从头部和腹部分离。 </li><li>用镊子打开胸腔检索珠。 注:珠子不移动到腹部，不与彼此接触，其中大部分在叔自由浮动他的血淋巴。要小心，不要中断与镊子珠，作为非珠白化可能很难找到。等待1〜5分钟，为珠，以获得颜色。 </li><li>转移珠在显微镜载玻片剥离液液滴，拍摄照片前。 注:无需洗珠。 </li><li>使用装备有相机的显微镜，采取各胎圈的数字图像在一个标准的照明设置和放大400倍。 注意:不要改变不同的图片之间的照明，使它们的比较。 </li></ol> 7.图像分析:珠黑化<ol><li>打开的图像分析软件。点击"文件"，然后点击"打开"，找到珠的形象。 </li><li>点击"分析"。点击"设置测量。"勾选在"测量设置"窗口中的"平均灰度值"框。 </li><li>点击圆圈选择工具图标。选择珠周长;留在方珠，避免周围的发光光环。 </li><li>点击"分析"菜单上。点击"措施"。获得胎圈的横截面面积的平均灰度值和大小。 <ol><li>要获得每个珠子的黑化值，使用计算器或计算器软件执行以下操作:"256 - 平均灰度值。"黑化的措施是0和256之间的值，其中0表示完全白色图像。 </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.

这种注射技术是很有用的激发和研究蚊子的黑化反应。例如，在这里我们研究的免疫刺激负荷的效果。 在此过程中的关键步骤是正确接种蚊子。以飞行肌或蚊子本身的任何过多的损害可能会阻止喂养蚊子或解剖之前，可能杀死它。第二个关键步骤是保持蚊子冰足够长的时间敲出来没有杀害他们。一个小容器将缓解和加快这一进程，因为它能够与冰更密切的联系。最后，在剥?...

昆虫依靠免疫反应，以保护自己免受寄生虫和致病菌1 - 3，通过他们的角质层或他们的肠上皮细胞4突破口。在蚊子，这些反应是对细菌5，6病毒，线虫丝虫7和疟原虫1,8,9高效。在蚊子，一个关键的免疫应答是外来颗粒与黑色素10的封装- 12。 12 -此封装可以在肠或血淋巴循环系统10发生。此黑化反应是亲酚的结果级联10 - 12，并且它可以导致寄生虫死亡或它们的吞噬作用。在成蚊，其中血细胞的细胞的数量是有限的，黑化是体液应答，如针对疟原虫寄生虫或丝虫线虫7。在其他一些昆虫，它直接是围绕寄生虫聚集melanize他们7血细胞的细胞。此外，黑色素也像鸡蛋的生产和表皮伤口愈合7其他几个生理过程是必不可少的。 免疫反应的刺激作为一种工具来研究昆虫干扰的几个农业和公共健康模型系统13 - 18。它在使用按蚊冈比亚蚊子，在非洲疟疾的主要载体，研究宿主-寄生虫的相互作用14 - 16,19。这些技术是基于昆虫以检测与它们的模式识别受体（PRR）2寄生虫的能力。蚊子也可以检测其他分子与其生物学如病原体相关分子模式（PAMP）的干扰，或检测由于胶原蛋白和核酸的释放自己的受损细胞。蚊子免疫细胞S，从而作为血细胞用于检测20 - 23。主免疫信号途径是Imd信号，收费，JAK / STAT 24和核糖核酸干扰（RNAi）25,26。这两种收费和IMD的途径影响黑色素的反应，并与亲酚互动级联10 - 12。 用于刺激黑色素响应的标准工具是用小珠进入胸腔的血淋巴蚊子的接种。黑色素包封的程度可以然后通过蚊子的解剖检索胎圈后测量19。在大多数研究中，只有一个小珠被每蚊15,16,27注入，但注入多珠是可能的，以研究在黑化响应19的限制。这些珠粒使用注射液（生理血清）来限制蚊子生理学紊乱注入和蚊子15,16,27的干燥。的染料加入到该溶液中，以缓解珠选择。它是用于检索珠子15,16,27解剖溶液是相同的。 用非致病性刺激接种昆虫的优点是把重点放在对免疫应答的直接作用的能力。有因寄生虫致病28，免疫抑制29没有复杂影响- 31，或免疫逃避31 - 34。此外，与其他生活史性状，如长寿或繁殖力的刺激所造成的后果，也可以研究。因此，研究人员在研究进化生态学可能需要这样的工具2,35,36。例如，免疫挑战黄蜂有饥饿状态下寿命缩短。免疫刺激和部署类似的负面影响已经在不同的无脊椎动物模型中观察到，往往导致在很短的呃寿命或更少的繁殖成功13,27,37。这些研究可以在不同的环境中2,4,38进行。刺激免疫力也感兴趣的那些直接专注于免疫病理学39,40。 这个协议是基于珠子的接种的蚊子刺激黑化反应，并直接测量黑色素的量。这使得在不同的实验设置黑化反应的定量和定性研究。这样的工具可以扩展到其它的免疫反应，如抗菌响应于刺激热灭活细菌41。它也可以在许多生态设置进行。

<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

免疫反应的刺激是无脊椎动物研究的常用工具来检查的有效性和免疫力的机制。这种刺激是基于注射非致病性颗粒的成虫，如颗粒将被免疫系统检测，将诱导产生免疫效应的。在这里，我们专注于在蚊子冈比亚按蚊黑化反应的刺激。黑化反应的结果在外来颗粒和寄生虫与黑色素的暗层封装。为了刺激这个回应，蚊子接种使用微细玻璃管在胸腔珠。然后，24小时后，蚊子解剖检索珠。胎圈的黑化的程度是使用图像分析软件来测量。珠没有寄生虫的致病作用，或它们的容量来逃避或抑制免疫应答。这些注射是measu方式再免疫效果和免疫刺激对其他生活史特征，如孕卵或寿命的影响。这是不完全一样的直接研究宿主 - 寄生虫的相互作用，但它是一个有趣的工具来研究免疫和其进化生态学。

<html>蚊子没有所有melanize以同样的方式的珠，如一些珠粒少覆盖有黑色素比其他（ 图1）。事实上，一些珠子由于缺乏黑色素的仍然蓝色，但其他人却完全黑了（ 图1）。黑化值通过线性内插标准化为值0（这对应于蓝色和unmelanized珠）和100（对应于一个黑暗和严重白化珠）（ 图2a和2b）之间。这些值表示的珠粒黑色素的程度...

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.

接种<em&gt;冈比亚按蚊</em&gt;珠蚊子诱发和测量黑化免疫反应

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

Research

JoVE Journal

Immunology and Infection

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

7.9K 次观看.  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.

视频: 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.

测量细菌感染小鼠和免疫反应李斯特菌</em

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

科研

免疫与感染

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.

寄生蜂简介<em&gt;果蝇</em&gt;和Antiparasite的免疫反应的

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.

活体成像的鼠标使用2 - 光子显微镜的胸腺

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.

定量测定的免疫反应和睡眠模式<em&gt;果蝇</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.

呼吸爆发的响应为先天免疫健康的斑马鱼中的指标量化

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.

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

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.

口咽部气管内PAMP管理和支气管肺泡灌洗的利用率来评估小鼠宿主的免疫反应

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.

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

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.

脂多糖治疗小鼠肝脏免疫应答中白细胞的双光子活体成像

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.

一种评价金黄色葡萄球菌感染先天免疫反应的小鼠模型

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