感谢Kaz Nagaosa和Akiko Shiratsuchi的意见。

准备<ol><li> 新鲜葡萄汁琼脂平板的制备 <ol><li>加入100毫升水至4.4克琼脂，加热微波炉中的混合物溶解琼脂。 </li><li>向琼脂溶液中加入80 mL新鲜葡萄汁，5 mL乙酸和5 mL乙醇。 </li><li>用移液管将约1.5 mL的溶液倒入每个载玻片上，使其固化。 </li></ol></li><li> 6厘米培养皿琼脂糖板的制备 <ol><li>向0.5g琼脂糖中加入50mL水，并在微波炉中加热混合物以溶解琼脂糖。 </li><li>将大约2.5 mL琼脂糖溶液倒入带有移液管的6 cm盘中。 </li></ol></li></ol>第16期胚胎收集<ol><li>收集成年苍蝇，并将200只雌性和200只雄性添加到50mL锥形管中。 </li><li>将一盘新鲜的葡萄汁琼脂放在酵母中放入50 mL管中，并用海绵盖上页。 </li><li>在16°C的温度下孵育苍蝇2 - 3天。每天更换一次板。 </li><li>在25°C将苍蝇移至黑暗1 h。 </li><li>将旧板更换为新的，不含酵母。让苍蝇放鸡2小时。 </li><li>收集板并在16℃孵育26小时。 </li><li>用油漆刷将板从板上收集到含有0.2％（v / v）Triton X-100的1mL PBS中。 </li><li>用含有0.2％（v / v）Triton X-100的PBS洗涤胚胎两次。 </li><li>为了去除绒毛膜，向胚胎中加入1.2mL次氯酸钠溶液（2.2-3.4％Cl）3分钟。 </li><li>用含有0.2％（v / v）Triton X-100的PBS洗涤胚胎4次。 </li><li>将胚胎放置在6厘米盘的琼脂糖板上。在罗伯茨20所述的显微镜下拿起16号胚胎。用微量移液管在1.5 mL的Treff微量试管中收集大约50个胚胎。 </li></ol>准备胚胎细胞配给<ol><li>用150μLPBS清洗胚胎两次。 </li><li>在PBS中用200μL胶原酶（0.25％（w / v））存在下，在1.5mL微量试管和沉淀混合器中使胚胎均质30次。 </li><li>通过移液10次分散胚胎细胞，并将细胞悬液移至1.5mL管中。在37℃下在水浴中孵育细胞1分钟。重复此步骤两次。 </li><li>在1400×g下添加PBS的800μL细胞悬浮液，和离心机在4℃下5分钟。 </li><li>取出上清液，加入PBS中的200μL胰蛋白酶（0.25％（w / v））至细胞。通过移液分离胚胎细胞50次。 </li><li>用70μm细胞过滤器过滤细胞悬浮液。 </li><li>为了停止胰蛋白酶活性，向过滤的细胞悬浮液中加入40μL热灭活的胎牛血清。加入800μLPBS，4℃下以1400× g离心5分钟。 </li><li>去除超新星个nt，暂停沉淀的细胞在PBS中的200μL，和离心机在1400×g下在4℃下5分钟。 </li><li>去除上清液，并将沉淀的细胞悬浮在30μL的PBS中。 </li><li>将制备的细胞悬浮液装载在氨基丙基三乙氧基硅烷涂覆的玻璃载片上，并等待10 - 15分钟，直到细胞附着在载玻片上。 </li><li>用移液器移除玻璃载玻片上的剩余溶液。将60-70μL含有4％（w / v）PFA（多聚甲醛）的PBS悬浮在细胞上10-15分钟进行固定。 </li><li>取出固定溶液，并将玻璃载玻片放入PBS中超过1分钟。 </li></ol> 4.血细胞染色<ol><li> 用抗Croquemort抗体进行免疫染色 <ol><li>将含有0.2％（v / v）Triton X-100的PBS中的载玻片在甲醇中连续浸泡10分钟，然后在PBS中10分钟。 </li><li>在PBS中装载20μL的5％（v / v）全猪血清ng 0.2％（v / v）Triton X-100在细胞上进行阻断。在室温下孵育玻片20分钟。 </li><li>移除玻璃载片上的溶液中，并在含有0.2％PBS安装抗Croquemort抗血清19，21（0.1％（V / V））的20μL（V / V）的Triton X-100细胞上。在4℃下孵育载玻片过夜。 </li><li>将含有0.2％（v / v）Triton X-100的PBS中的载玻片浸泡10分钟5次。 </li><li>在PBS中浸泡玻片10分钟。 </li><li>在室内的细胞上装载含有0.2％（v / v）Triton X-100和5％（v / v）全猪血清的PBS中的20μL碱性磷酸酶标记的抗大鼠IgG（0.5％（v / v）温度1 h。 </li><li>将含有0.2％（v / v）Triton X-100的PBS中的载玻片浸泡10分钟5次。 </li><li>将含有100mM Tris-HCl，pH9.5,100mM NaCl和50mM MgCl 2的缓冲液中的载玻片浸泡10分钟。 </li><li>含有0.23mg / mL 5-溴-4的载体磷酸酶底物溶液（BCIP），0.35mg / mL硝基蓝四唑（NBT），100mM Tris-HCl，pH9.9,100mM NaCl和50mM MgCl 2 。 </li><li>在显微镜下观察细胞是否正常染色。当血细胞颗粒中出现强烈的紫色信号时，取出底物溶液，并将载玻片浸泡在由10mM Tris-HCl，pH8.5和1mM EDTA组成的缓冲液中。建议染色约5 - 10分钟。 </li></ol></li><li> 用抗GFP抗体进行免疫染色（血细胞染色的另一种选择） <ol><li>将载玻片在甲醇中连续浸泡10分钟，含有0.2％（v / v）Triton X-100的PBS 10分钟，PBS 10分钟。 </li><li>将含有0.2％（v / v）Triton X-100的PBS中的5％（v / v）全猪血清装入20μL用于阻断的细胞上。在室温下孵育玻片20分钟。 </li><li>取出载玻片上的溶液，安装20μL小鼠抗GFP抗体（1％（v / v））/ PBS c在细胞上培养0.2％（v / v）Triton X-100。在室温下孵育玻片一夜。 </li><li>将含有0.2％（v / v）Triton X-100的PBS中的载玻片浸泡10分钟5次。 </li><li>在PBS中浸泡玻片10分钟。 </li><li>在室内的细胞上装载含有0.2％（v / v）Triton X-100和5％（v / v）全猪血清的PBS中的20μL碱性磷酸酶标记的抗小鼠IgG（0.5％（v / v）温度1 h。 </li><li>将含有0.2％（v / v）Triton X-100的PBS中的载玻片浸泡10分钟5次。 </li><li>将载玻片在100mM Tris-HCl，pH9.9,100mM NaCl和50mM MgCl 2组成的缓冲液中浸泡10分钟。 </li><li>将含有0.23mg / mL BCIP，0.35mg / mL NBT，100mM Tris-HCl，pH9.9,100mM NaCl和50mM MgCl 2的磷酸酶底物溶液装载在细胞上。 </li><li>在显微镜下观察细胞是否正常染色。当紫色信号出现在全血细胞中时，取出底物溶液，并将缓冲液中的载玻片浸泡10mM Tris-HCl，pH8.5和1mM EDTA。建议染色约10 - 15分钟。 </li></ol></li></ol> 5. TUNEL染色细胞凋亡细胞<ol><li>将PBS在PBS中浸泡5分钟。 </li><li>用新的PBS重复。 </li><li>将平衡缓冲液（见材料表）放在细胞上10分钟。 </li><li>从玻片上取出溶液，并在37℃下将20μL含有5μL末端脱氧核苷酸转移酶的TdT溶液和15μL反应缓冲液装入细胞1小时。 </li><li>从玻片上取出溶液，并将缓冲液浸入缓冲液中，缓冲液由0.5 mL STOP / Wash Buffer和17 mL水组成。 </li><li>在PBS中浸泡载玻片5分钟3次。 </li><li>在室温下将20μL抗地高辛 - 过氧化物酶载于细胞上30分钟。 </li><li>将载玻片在PBS中浸泡5分钟4次。 </li><li>将载玻片浸泡在30mL含有pH7.5的50mM Tris-HCl的过氧化物酶底物溶液中3,9&#39;-二氨基联苯胺四氯化氢（DAB）和0.002％（v / v）H 2 O 2的全微微铲30秒。 </li><li>重复步骤5.9直到凋亡细胞染成棕色。将水玻片浸泡在水中以停止过氧化物酶反应。 </li><li>用水封闭细胞观察。 </li></ol> 6.测量凋亡细胞的吞噬水平<ol><li>观察样品在光学显微镜下，以评估吞噬的水平。计数Croquemort阳性细胞或GFP阳性细胞作为血细胞，TUNEL阳性细胞作为凋亡细胞，双重阳性细胞作为吞噬血细胞。 </li><li>吞噬指数定义为双重阳性细胞数与Croquemort阳性细胞或GFP阳性细胞总数的数量。建议观察超过300个血细胞。 </li></ol>

<ol>
	<li>Danial, N. N., Korsmeyer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death:+critical+control+points.">Cell death: critical control points.</a> Cell. 116 (2), 205-219 (2004).</li><li>Vaux, D. L., Korsmeyer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+in+development.">Cell death in development.</a> Cell. 96 (2), 245-254 (1999).</li><li>Savill, J., Fadok, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corpse+clearance+defines+the+meaning+of+cell+death.">Corpse clearance defines the meaning of cell death.</a> Nature. 407 (6805), 784-788 (2000).</li><li>Lauber, K., Blumenthal, S. G., Waibel, M., Wesselborg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clearance+of+apoptotic+cells:+getting+rid+of+the+corpses.">Clearance of apoptotic cells: getting rid of the corpses.</a> Mol Cell. 14 (3), 277-287 (2004).</li><li>Ravichandran, K. S., Lorenz, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engulfment+of+apoptotic+cells:+signals+for+a+good+meal.">Engulfment of apoptotic cells: signals for a good meal.</a> Nat Rev Immunol. 7 (12), 964-974 (2007).</li><li>Ravichandran, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beginnings+of+a+good+apoptotic+meal:+the+find-me+and+eat-me+signaling+pathways.">Beginnings of a good apoptotic meal: the find-me and eat-me signaling pathways.</a> Immunity. 35 (4), 445-455 (2011).</li><li>Nakanishi, Y., Nagaosa, K., Shiratsuchi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytic+removal+of+cells+that+have+become+unwanted:+implications+for+animal+development+and+tissue+homeostasis.">Phagocytic removal of cells that have become unwanted: implications for animal development and tissue homeostasis.</a> Dev Growth Differ. 53 (2), 149-160 (2011).</li><li>Reddien, P. W., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+engulfment+process+of+programmed+cell+death+in+caenorhabditis+elegans.">The engulfment process of programmed cell death in caenorhabditis elegans.</a> Annu Rev Cell Dev Biol. 20, 193-221 (2004).</li><li>Hedgecock, E. M., Sulston, J. E., Thomson, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+affecting+programmed+cell+deaths+in+the+nematode+Caenorhabditis+elegans.">Mutations affecting programmed cell deaths in the nematode Caenorhabditis elegans.</a> Science. 220 (4603), 1277-1279 (1983).</li><li>Ellis, R. E., Jacobson, D. M., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+required+for+the+engulfment+of+cell+corpses+during+programmed+cell+death+in+Caenorhabditis+elegans.">Genes required for the engulfment of cell corpses during programmed cell death in Caenorhabditis elegans.</a> Genetics. 129 (1), 79-94 (1991).</li><li>Gumienny, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CED-12/ELMO,+a+novel+member+of+the+CrkII/Dock180/Rac+pathway,+is+required+for+phagocytosis+and+cell+migration.">CED-12/ELMO, a novel member of the CrkII/Dock180/Rac pathway, is required for phagocytosis and cell migration.</a> Cell. 107 (1), 27-41 (2001).</li><li>Hsu, T. Y., Wu, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engulfment+of+apoptotic+cells+in+C.+elegans+is+mediated+by+integrin+alpha/SRC+signaling.">Engulfment of apoptotic cells in C. elegans is mediated by integrin alpha/SRC signaling.</a> Curr Biol. 20 (6), 477-486 (2010).</li><li>Hanayama, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+factor+that+links+apoptotic+cells+to+phagocytes.">Identification of a factor that links apoptotic cells to phagocytes.</a> Nature. 417 (6885), 182-187 (2002).</li><li>Tepass, U., Fessler, L. I., Aziz, A., Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+origin+of+hemocytes+and+their+relationship+to+cell+death+in+Drosophila.">Embryonic origin of hemocytes and their relationship to cell death in Drosophila.</a> Development. 120 (7), 1829-1837 (1994).</li><li>Gold, K. S., Bruckner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+and+cellular+immunity+in+Drosophila+melanogaster.">Macrophages and cellular immunity in Drosophila melanogaster.</a> Semin Immunol. 27, 357-368 (2015).</li><li>Abrams, J. M., White, K., Fessler, L. I., Steller, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmed+cell+death+during+Drosophila+embryogenesis.">Programmed cell death during Drosophila embryogenesis.</a> Development. 117 (1), 29-43 (1993).</li><li>Franc, N. C., Heitzler, P., Ezekowitz, R. A., White, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+for+croquemort+in+phagocytosis+of+apoptotic+cells+in+Drosophila.">Requirement for croquemort in phagocytosis of apoptotic cells in Drosophila.</a> Science. 284 (5422), 1991-1994 (1999).</li><li>Mukae, N., Yokoyama, H., Yokokura, T., Sakoyama, Y., Nagata, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+innate+immunity+in+Drosophila+by+endogenous+chromosomal+DNA+that+escaped+apoptotic+degradation.">Activation of the innate immunity in Drosophila by endogenous chromosomal DNA that escaped apoptotic degradation.</a> Genes Dev. 16 (20), 2662-2671 (2002).</li><li>Manaka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Draper-mediated+and+phosphatidylserine-independent+phagocytosis+of+apoptotic+cells+by+Drosophila+hemocytes/macrophages.">Draper-mediated and phosphatidylserine-independent phagocytosis of apoptotic cells by Drosophila hemocytes/macrophages.</a> J Biol Chem. 279 (46), 48466-48476 (2004).</li><li>Roberts, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila: A Practical Approach. , 3868-3878 (1998).</li><li>Kuraishi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pretaporter,+a+Drosophila+protein+serving+as+a+ligand+for+Draper+in+the+phagocytosis+of+apoptotic+cells.">Pretaporter, a Drosophila protein serving as a ligand for Draper in the phagocytosis of apoptotic cells.</a> Embo j. 28 (24), 3868-3878 (2009).</li><li>Franc, N. C., Dimarcq, J. L., Lagueux, M., Hoffmann, J., Ezekowitz, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Croquemort,+a+novel+Drosophila+hemocyte/macrophage+receptor+that+recognizes+apoptotic+cells.">Croquemort, a novel Drosophila hemocyte/macrophage receptor that recognizes apoptotic cells.</a> Immunity. 4 (5), 431-443 (1996).</li><li>Kuraishi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+calreticulin+as+a+marker+for+phagocytosis+of+apoptotic+cells+in+Drosophila.">Identification of calreticulin as a marker for phagocytosis of apoptotic cells in Drosophila.</a> Exp Cell Res. 313 (3), 500-510 (2007).</li><li>Nagaosa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+betanu-mediated+phagocytosis+of+apoptotic+cells+in+Drosophila+embryos.">Integrin betanu-mediated phagocytosis of apoptotic cells in Drosophila embryos.</a> J Biol Chem. 286 (29), 25770-25777 (2011).</li><li>Nonaka, S., Nagaosa, K., Mori, T., Shiratsuchi, A., Nakanishi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+alphaPS3/betanu-mediated+phagocytosis+of+apoptotic+cells+and+bacteria+in+Drosophila.">Integrin alphaPS3/betanu-mediated phagocytosis of apoptotic cells and bacteria in Drosophila.</a> J Biol Chem. 288 (15), 10374-10380 (2013).</li><li>Fujita, Y., Nagaosa, K., Shiratsuchi, A., Nakanishi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+NPxY+motif+in+Draper-mediated+apoptotic+cell+clearance+in+Drosophila.">Role of NPxY motif in Draper-mediated apoptotic cell clearance in Drosophila.</a> Drug Discov Ther. 6 (6), 291-297 (2012).</li><li>Bruckner, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PDGF/VEGF+receptor+controls+blood+cell+survival+in+Drosophila.">The PDGF/VEGF receptor controls blood cell survival in Drosophila.</a> Dev Cell. 7 (1), 73-84 (2004).</li><li>Nonaka, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+Pathway+for+Phagocyte+Priming+upon+Encounter+with+Apoptotic+Cells.">Signaling Pathway for Phagocyte Priming upon Encounter with Apoptotic Cells.</a> J Biol Chem. 292 (19), 8059-8072 (2017).</li><li>Hanayama, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+disease+and+impaired+uptake+of+apoptotic+cells+in+MFG-E8-deficient+mice.">Autoimmune disease and impaired uptake of apoptotic cells in MFG-E8-deficient mice.</a> Science. 304 (5674), 1147-1150 (2004).</li></ol>

作者宣称他们没有竞争的经济利益。

<html>我们在此描述了使用果蝇胚胎的吞噬作用。通过使用分散的胚胎细胞定量测量吞噬作用，将血细胞， 果蝇专业吞噬细胞免疫染色用于血细胞标记Croquemort或srpHemo-驱动的GFP，并且在该方案中通过TUNEL检测凋亡细胞。吞噬水平通过计数血细胞总数和吞噬血细胞来表达为吞噬指数。使用分散的胚胎细胞使我们能够观察全胚胎中的所有吞噬细胞和凋亡细胞，并精确量化吞噬水平。?...

在后生动物， 如 线虫 ，果蝇果蝇和小鼠和人类，大量细胞的发育过程中发生凋亡来塑造自己的身体，并在成年后保持动态平衡1，2。凋亡细胞需要快速去除，因为它们通过释放免疫原性的细胞内物质而在周围组织中引起炎症3 。为了便于快速去除，本凋亡细胞所谓吃我信号由吞噬细胞的吞噬受体识别，并通过吞噬作用3，4，5，6被消除。因此，吞噬作用在维持宿主体内平衡方面起关键作用，因此阐明了分子<html>凋亡细胞吞噬的潜在机制是重要的。 负责凋亡细胞的吞噬功能的机制似乎在线虫，果蝇和小鼠7种之中进化上保守的。目前可以使用几种吞噬作用来评估这些模型动物中凋亡细胞的吞噬。在秀丽隐杆线虫中 ，131个体细胞在发育期间经历程序性细胞死亡，细胞尸体被非专业吞噬细胞的相邻细胞吞噬8 。因此，计数线虫中剩余细胞尸体的数量表示体内吞噬水平。通过搜索线虫突变体，显示死细胞数量的增加，吞噬作用所需的几个基因已被鉴定和基因表征9，10，S ="外部参照"&gt; 11,12。 原代培养吞噬细胞（通常是巨噬细胞）的离体吞噬作用常常用于小鼠。使用细胞系如Jurkat细胞制备凋亡细胞，并与原代吞噬细胞混合。孵育数小时后，计数吞噬细胞和吞噬吞噬细胞的总数，以评估吞噬水平。作为这种方法的一个复杂的变形例中，永田的小组开发的体外吞噬作用测定与细胞中表达一个抗胱天蛋白酶ICAD（抑制剂胱天蛋白酶活化的DNA酶），其中凋亡细胞不进行细胞凋亡的DNA片段化，但DNA仍裂解时细胞被吞噬。当这些细胞在吞噬作用测定中用作凋亡靶点时，只有吞噬凋亡细胞的DNA被破碎，并通过TdT介导的dUTP缺口末端标记（TUNEL）染色。所以，水稻通过在吞噬细胞和凋亡细胞的混合物中计数TUNEL信号来测量凋亡细胞吞噬13 。 在果蝇 ，名为血细胞，巨噬细胞果蝇 ，专业的吞噬细胞负责细胞凋亡14，15的吞噬作用。除了与培养的细胞系在体外吞噬作用测定法， 体内用全果蝇胚胎的吞噬作用测定法是可用的。 果蝇胚胎是用于检查凋亡细胞吞噬的水平，因为许多细胞发生凋亡和胚胎发育14，15，16时由血细胞吞噬的有力工具。 体内吞噬试验的一个实例是法国集团开发的方法。在他们的方法中，血细胞是de通过免疫染色过氧化物酶，血细胞标志物，凋亡细胞用核染料，7-氨基放线菌素D在整个果蝇胚胎中染色，双阳性细胞数被计为吞噬信号17 。对胚胎的吞噬作用测定的另一个例子是基于上述Nagata方法的概念;然而， 在体内的吞噬作用是使用DCAD的胚胎进行评估（ 果蝇胱天蛋白酶活化的DNA酶）突变体蝇18,19。这些体内吞噬试验可用于原位直接观察吞噬作用。然而，难以排除计数吞噬细胞步骤中的任何可能的偏差，因为由于其厚度难以观察全胚胎中的所有吞噬细胞和凋亡细胞。 为了克服这个限制，我们开发了在果蝇胚胎中进行新的吞噬作用测定。在我们的方法中，为了容易地计数吞噬血细胞，将整个胚胎均匀化以制备分散的胚胎细胞。通过吞噬细胞标志物的免疫染色检测吞噬细胞，并用这些分散的胚胎细胞用TUNEL检测凋亡细胞。使用分散的胚胎细胞使我们能够测量体内吞噬作用水平，好像我们进行了精确量化吞噬水平的体外吞噬作用。如果它们发展到胚胎20的第16阶段，则吞噬作用的凋亡细胞清除率最丰富的发育阶段，则可以在该测定中使用所有基因型的苍蝇。该方法具有定量评估吞噬水平的优点，因此有助于鉴定参与体内凋亡细胞吞噬的新分子。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>whole swine serum</td>
			<td>MP Biomedicals</td>
			<td>55993</td>
			<td>For bloking</td>
		</tr>
		<tr>
			<td>Treff micro test tube(easy fit)&#160; Dnase, Rnase free tube, 1.5 mL</td>
			<td>TreffLab</td>
			<td>96. 4625. 9. 01</td>
			<td>For&#160; homogenization</td>
		</tr>
		<tr>
			<td>pellet mixer&#160; 1.5 mL</td>
			<td>TreffLab</td>
			<td>96. 7339. 9. 03</td>
			<td>For&#160; homogenization</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Sigma-Aldrich</td>
			<td>C-0130</td>
			<td>For preparation of embryonic cells</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Thermo Fisher SCIENTIFIC</td>
			<td>27250-018</td>
			<td>For preparation of embryonic cells</td>
		</tr>
		<tr>
			<td>Kpl Anti-Rat IgG (H+L) Ab MSA, AP</td>
			<td>KPL</td>
			<td>475-1612</td>
			<td>secondary antibody for 
			stainig hemocytes with an anti-Croquemort antibody</td>
		</tr>
		<tr>
			<td>5-bromo-4-chloro- 
			3-indolyl-phosphate</td>
			<td>Roche</td>
			<td>11383221001</td>
			<td>BCIP, For staining of hemocytes</td>
		</tr>
		<tr>
			<td>nitro blue tetrazolium</td>
			<td>Roche</td>
			<td>11383213001</td>
			<td>NBT, For staining of hemocytes</td>
		</tr>
		<tr>
			<td>Anti-Croquemort antibody</td>
			<td></td>
			<td></td>
			<td>described previously in Manaka et al, J. Biol. Chem., 279, 48466-48476</td>
		</tr>
		<tr>
			<td>&#160;Anti-GFP 
			from mouse IgG1&#954; (clones 7.1 and 13.1)&#160;</td>
			<td>Roche</td>
			<td>11814460001</td>
			<td>For staining of hemocytes</td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG-AP Conjugate</td>
			<td>Bio-Rad</td>
			<td>170-6520</td>
			<td>secondary antibody for stainig hemocytes with an anti-Croquemort antibody</td>
		</tr>
		<tr>
			<td>Apop Tag Peroxidase In Situ Apoptosis Detection Kit</td>
			<td>Millipore</td>
			<td>S7100</td>
			<td>For staining of apoptoitc cells. This kit includes Equilibration buffer, Reaction buffer, STOP/Wash buffer, TdT enzyme, and Anti-Digoxigenin-Peroxidase.</td>
		</tr>
		<tr>
			<td>3,3&#39;-diaminobenzidine 
			tetrahydrichloride</td>
			<td>nacalai tesque</td>
			<td>11009-41</td>
			<td>DAB, For staining of apoptoitc cells</td>
		</tr>
		<tr>
			<td colspan="2">Table of Fly Strains</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>w1118</td>
			<td></td>
			<td></td>
			<td>Control flies, described in Freeman et al., Neuron, 38, 567-580</td>
		</tr>
		<tr>
			<td>drpr&#916;5</td>
			<td></td>
			<td></td>
			<td>drpr mutant, described in Freeman et al, Neuron, 38, 567-580</td>
		</tr>
		<tr>
			<td>Itgbn2</td>
			<td></td>
			<td></td>
			<td>Itgbn mutant, described in Devenport et al., Development, 131, 5405-5415</td>
		</tr>
		<tr>
			<td>srpHemoGAL4 UAS-EGFP</td>
			<td></td>
			<td></td>
			<td>described in Br&#252;ckner et al., Dev. Cell., 7, 73-84</td>
		</tr>
		<tr>
			<td>UAS-drpr-IR</td>
			<td>VDRC</td>
			<td>4833</td>
			<td>-</td>
		</tr>
		<tr>
			<td>UAS-Itgbn-IR</td>
			<td>NIG-fly</td>
			<td>1762R-1</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

phagocytosis assay for apoptotic cells in drosophila embryos

由于其在免疫和炎症难治性疾病中的作用，需要更详细地阐明凋亡细胞吞噬作用的分子机制。我们本文开发了一种使用果蝇果蝇定量研究吞噬作用的实验方法，其中基因网络控制吞噬反应是从哺乳动物进化保守的。为了准确地检测和计数使用全部动物吞噬和吞噬吞噬细胞，将果蝇胚胎匀浆以获得包括吞噬细胞和凋亡细胞的分散细胞。使用分散的胚胎细胞使我们能够测量体内吞噬水平，就像我们进行体外吞噬作用测定一样，可以观察全胚胎中的所有吞噬细胞和凋亡细胞，并精确定量吞噬水平。我们确认这种方法再现了以前的研究确定了凋亡细胞吞噬所需的基因。这种方法允许吞噬死细胞，当与强大的果蝇遗传学结合时，将揭示吞噬细胞凋亡细胞的迁移，识别，吞噬和降解所引起的复杂的吞噬反应。

为了检测凋亡细胞的吞噬作用，收集了发育阶段16的果蝇胚胎，并作为分散细胞制备。血细胞，巨噬细胞果蝇 ，进行免疫细胞化学对血细胞标记"Croquemort" 17，22使用特异性抗体19,21染色，凋亡细胞通过TUNEL在分散的胚胎细胞（ 图1A）染色。 Croctemort，?...

Watch this Scientific Journal Video about Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos at JoVE.com

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

我们在此描述使用果蝇分散的胚胎细胞的吞噬作用。它使我们能够容易且准确地量化体内吞噬水平，并确定凋亡细胞吞噬所需的新分子。

细胞凋亡细胞的吞噬作用<em&gt;果蝇</em&gt;胚胎

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and ...

phagocytosis-assay-for-apoptotic-cells-in-drosophila-embryos

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Immunology and Infection

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

Unit 1

Cell Death

SCIENTISTS IN ACTION

9.8K 次观看.  Kanazawa University. 我们在此描述使用果蝇分散的胚胎细胞的吞噬作用。它使我们能够容易且准确地量化体内吞噬水平，并确定凋亡细胞吞噬所需的新分子。 

视频: Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

RNAi筛选参与宿主因素牛痘病毒感染使用果蝇细胞

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

科研

免疫与感染

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

使用双光子显微镜在受感染的肺部的嗜中性粒细胞吞噬和NET形成的直接观察

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

量<em在体外</em&gt;激活的主人微血管内皮细胞在静态条件下测量的检测，以中性粒细胞粘附

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an intricate network of inducers, detectors, and activators rapidly responds to neuron damage, infection or other immune infractions. This inflammation of immune cells is intimately associated with the pathology of neurodegenerative disorders, such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease and ALS. Under compromised disease states, chronic inflammation, intended to minimize neuron damage, may lead to an over-excitation of the immune cells, ultimately resulting in the exacerbation of disease progression. For example, loss of dopaminergic neurons in the midbrain, a hallmark of PD, is accelerated by the excessive activation of the inflammatory response. Though the cause of PD is largely unknown, exposure to environmental toxins has been implicated in the onset of sporadic cases. The herbicide paraquat, for example, has been shown to induce Parkinsonian-like pathology in several animal models, including Drosophila melanogaster. Here, we have used the conserved innate immune response in Drosophila to develop an assay capable of detecting varying levels of nitric oxide, a cell-signaling molecule critical to the activation of the inflammatory response cascade and targeted neuron death. Using paraquat-induced neuronal damage, we assess the impact of these immune insults on neuroinflammatory stimulation through the use of a novel, quantitative assay. Whole brains are fully extracted from flies either exposed to neurotoxins or of genotypes that elevate susceptibility to neurodegeneration then incubated in cell-culture media. Then, using the principles of the Griess reagent reaction, we are able to detect minor changes in the secretion of nitric oxide into cell-culture media, essentially creating a primary live-tissue model in a simple procedure. The utility of this model is amplified by the robust genetic and molecular complexity of Drosophila melanogaster, and this assay can be modified to be applicable to other Drosophila tissues or even other small, whole-organism inflammation models.

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Levels of the inflammatory cell-signaling molecule nitric oxide (NO) are commonly assayed using Griess reagent. In this protocol, we have created a modified Griess assay utilizing live Drosophila brain tissue in order to detect the secretion of NO in a simple, quantifiable and highly repeatable method.

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an ...

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

猪或牛光感受器外段的细胞吞噬分析对视网膜色素上皮细胞大规模纯化

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

单核细胞单层试验中用于评估Fcγ受体介导的吞噬作用

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

人干扰素α超灵敏单分子阵列数字酶联免疫吸附试验的研制与验证

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the removal of dead cells or foreign particles, and in the resolution of inflammatory responses and tissue remodeling, processes that are mediated by various surface receptors of the AMs. Here, we report methods for the analysis of the phagocytic function of AMs using in vitro and in vivo assays and experimental strategies to differentiate between the pattern recognition receptor-, complement receptor-, and Fc gamma receptor-mediated phagocytosis. Finally, we discuss a method to establish and characterize a P. aeruginosa pneumonia model in mice to assess bacterial clearance in vivo. These assays represent the most common methods to evaluate AM functions and can also be used to study macrophage function and bacterial clearance in other organs.

Here we report common methods to analyze the phagocytic function of murine alveolar macrophages and bacterial clearance from the lung. These methods study in vitro phagocytosis of fluorescein isothiocyanate beads and in vivo phagocytosis of Pseudomonas aeruginosa Green Fluorescent Protein. We also describe a method for clearing P. aeruginosa in mice.

肺泡巨噬细胞吞噬作用与小鼠细菌清除

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the ...

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

果蝇、果蝇细胞免疫应答的评估

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...