The authors are financially supported by the Swiss National Science Foundation through an operating grant N&#176; 31003A-149566 (to N.D.), and The Sir Jules Thorn Charitable Overseas Trust through a Young Investigator Subsidy (to P.N.).

伦理声明：所有的动物操作均严格按照日内瓦大学的动物研究委员会的指导方针进行。 1.准备吞噬目标<ol><li>添加20毫克干燥酵母多糖至10毫升无菌磷酸盐缓冲盐水（PBS）中。涡流和热在沸水浴10分钟。凉爽和离心机在2000×g离心5分钟。 </li><li>除去上清液，重悬在1ml PBS中并声处理用于在水浴超声波仪10分钟。转移500μl到2个1.5 mL管。离心在11000×g离心5分钟。 </li><li>重复用500微升PBS洗涤。煮沸酵母多糖可以等分，冷冻并储存在-20℃。 </li><li>于500μl新鲜制备的0.1M 的 Na 2 CO 3，pH值9.3如上述（步骤1.2）洗两次酵母聚糖。 </li><li>悬浮在最后一次洗涤后490微升。添加10微升10毫克/毫升的FITC溶于二甲sulfoxidE（DMSO）中，涡旋，盖上箔和孵育在搅拌4小时，在室温。 </li><li>去除未结合的染料，与上述（步骤1.2）洗涤，先用0.5毫升DMSO + 150微升的PBS，然后0.5毫升DMSO + 300微升的PBS，然后0.5毫升DMSO + 450微升PBS中，然后单独的PBS。 </li><li>使用血球计数酵母聚糖。加入1μl酵母多糖的500微升PBS中，涡旋，然后加10微升到血细胞计数器室中，在那里它满足盖玻片放置在中央网格的边缘。装入血球上一个明亮的视野显微镜配备了20倍的目标。 </li><li>专注于含有16平方上的左上角和计数用手握式计数器在这一领域的所有酵母多糖颗粒。重复与较低的右上角。计算使用下面的等式的酵母聚糖浓度：酵母聚糖浓度=计数/ 2×500×10 4的酵母多糖/毫升。标记的酵母多糖可以等分，冷冻并储存在-20℃。 </li><li> Opsonize标记的酵母多糖无线TH兔抗酵母聚糖抗体以1：100的稀释。涡孵育1小时在37℃水浴。用500μlPBS洗涤如上（步骤1.2）。 注：超声强烈建议，特别是调理后，因为酵母多糖往往形成团块，可以使分析更困难以后。调理作用用65％体积/体积的大鼠或人血清可以用作替代方法。 </li><li>计数使用血球酵母聚糖如步骤1.7-1.8中描述，并稀释至100×10 6个颗粒/毫升。调理作用与其他吞噬刺激器，如补体或无调理作用，可以替代地执行。 </li></ol> 2.实时视频显微镜<ol><li>如其他地方38中详细描述隔离鼠标主中性粒细胞。 <ol><li>另外，使用任何吞噬细胞类型。保持在冰上的中性粒细胞在罗斯韦尔园区纪念研究所（RPMI）1640培养基中补充有5％胎牛血清（体积/体积），200单位青霉素/ streptomyciN，以10×10 6细胞/ ml的浓度，直到准备使用。其它细胞类型可被1-4天前接种。 </li></ol></li><li>加入2×10 5（20微升）的嗜中性粒细胞，以35毫米玻璃底皿的中心，通过加入50μl培养基的传播降和孵育在37℃下5分钟，以使细胞粘附。 </li><li>加入1毫升的Hank氏平衡盐溶液（HBSS）加热到37℃，含有特定的MPO抑制剂4-氨基肼（4- ABH，10μM）。非特异性的MPO抑制剂，如叠氮化钠（5毫摩），也可以使用，但最近已建议施加于pH值39附加的非特异性作用。 </li><li>安装在配有37℃的供暖系统和40X油目标的一个宽视场荧光显微镜的菜。孵育细胞无成象10分钟，以允许细胞和设备达到平衡。 </li><li>调整显微镜设置以获得一个亮场图像传输[PHA本身或微分干涉对比（DIC）如果可用〕用440纳米或490纳米激发和535纳米的发射，每30秒。 </li><li>根据显微镜系统，以避免过多的光漂白和光毒性，并根据实验问题被要求调整曝光时间和采集频率。开始图像采集。 </li><li> 2分钟后，加入10×10 6（50微升）调理的FITC的酵母聚糖的培养皿的中心。调整目标：细胞比率取决于所用吞噬细胞和目标的类型。 </li><li>继续成像30分钟。 30分钟后，停止时间推移采集和启动一个定时器。移动载物台并捕获10个或更多的快照在视图像其他吞噬体不同的领域中，所有在5分钟内。 </li></ol> 3.校准和控制实验<ol><li>制备pH校准溶液（配方在表1中记载 ）之前实验当天和冻结在10ml等分试样。解冻校准解决方案和温暖至37℃的水浴中。检查用PH计的pH值，并记录溶液的实际pH值。 </li><li>安装在玻璃底菜之前，图像采集蠕动泵及以上的（第2节）所描述的现场表演视频显微镜。 </li><li>在30分钟采集期间结束打开以除去培养皿内的溶液泵，加入1毫升第一校准溶液和停止泵。 </li><li>等到信号稳定，一般为5分钟。重复每个校准解决方案。 <ol><li>执行每个实验每天至少一个校准和校准开始具有最高pH值和工作顺序至最低，以及从最低pH值开始，并依次朝向最高工作。 </li></ol></li><li>作为对照，添加100微升100μM的NADPH氧化酶抑制剂二苯propionium碘（DPI）稀释于HBSS中的单元格900微升HBSS在玻璃底皿中，孵育10分钟。 </li><li>开始收购并添加酵母多糖如上面（步骤2.7）。吞噬后15分钟，加入10微升10μMV-ATP酶抑制剂concanamycin A（CONCA）稀释于HBSS，并继续成像额外的15分钟。 </li></ol> 4.分析<ol><li>分析时间推移电影和吞噬快照。 注：以下说明是特定的ImageJ的。一步一步的说明可能很大程度上取决于所使用的软件，但在本节中描述的功能是最专业图像分析软件。 <ol><li>打开与专业图像分析软件的映像。在那里有没有细胞的区域绘制一个小广场的投资回报率与方多边形选择工具，然后单击从投资回报率插件&gt; BG减法减去了490通道的背景。然后上传的440路相同的投资回报率通过单击编辑&gt;选项&gt;恢复选择并重复。 </li><li>使490路的440路划分的32位比图像通过单击流程&gt;图像计算器...，选择在Image1的和IMAGE2下拉菜单中相应的图像，然后选择在操作下拉菜单中选择“鸿沟” ，并检查了32位（浮点）结果复选框。 </li><li>点击图片&gt;查找表&gt;彩虹RGB改变颜色编码查表到比兼容的彩色编码。阈值的比例进行图像消除0到无穷大的像素通过点击图像&gt;调整&gt;阈值...。调整滚动条，使酵母多糖显示为红色，点击Apply，确保设置背景像素为NaN复选框被选中。 </li><li>点击图片结合这个比例堆栈明视场通道成多路复合&gt;颜色&gt;合并通道，并选择在灰色通道下拉菜单中的亮场图像，并在绿色通道下拉菜单中的比例图像，选择保持源图像复选框。扫描时间推移电影或快照，以确定哪些吞噬体是待分析。明视场通道是这个有用的。 </li><li>通过绘制吞噬体周围的椭圆形的多边形选择工具感兴趣区域（ROI），并将其通过单击分析&gt;工具&gt;投资回报率管理器添加到投资回报率经理&gt;添加。点击更多&gt;多功能测量和去选择每片复选框的一行衡量他们的酵母多糖强度比。 </li><li>对于时间推移电影，按部就班地进行绘制的投资回报率，按Ctrl + M来衡量每一个时间点，而移动的投资回报率在必要时随着时间的推移跟踪强度值吞噬体一次。从结果窗口中的测量结果复制到一个电子表格软件。 注意：独立实验15（每实验×3 5时程）分析是理想的，但或多或​​少可根据观察到的变异性是必需的。保存的投资回报总是建议。有些软件包允许图像REGI共探和投资回报的动态更新，有利于这部分的分析。 </li></ol></li><li>转换，从荧光至pH值<ol><li>测量的第4.1节所述的四百四十分之四百九十○比例在校准时间推移电影吞噬体。 </li><li>在电子表格中，绘制比值随着时间的推移，而从视场之内的所有吞​​噬体5的时间点的时间段对应于每个pH校正中间内计算平均比率值（通常5-20之间）解。 （见红色框图5A）。 </li><li>暗算实际测得的pH值，这些平均四百四十○分之四百九十零比值。结合至少3个独立校准到一个单一的图形。使用统计数值或软件包执行非线性回归（最佳拟合曲线），通常以S形方程。 注意：例如，在图5B中使用的方程为一个玻尔兹曼S形[Y =底+（（顶-底）/（1 + EXP（（V50-X）/斜率））]，其中Y值是四百四十〇分之四百九十〇比率中，X是将pH值和顶部，底部，V50和斜率参数由软件来计算。 </li><li>使用所获得的公式来转换比的值至pH。 注意：例如，在图5B中的叠氮化钠存在下，校准曲线得到的方程是四百四十○分之四百九十○比率= 1.103 +（2.89-1.103）/（1 + EXP（（6.993-pH值）/0.9291）] 。求解用于pH给出以下等式：pH值= 6.993 - 0.9291 * [LN（（1.178 /（比-1.103）） - 1））]。 </li></ol></li></ol>

<ol>
	<li>Yeung, T., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+signaling+and+the+modulation+of+surface+charge+during+phagocytosis.">Lipid signaling and the modulation of surface charge during phagocytosis.</a> Immunol Rev. 219, 17-36 (2007).</li><li>Flannagan, R. S., Jaumouille, V., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+phagocytosis.">The cell biology of phagocytosis.</a> Annu Rev Pathol. 7, 61-98 (2012).</li><li>Fairn, G. D., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+nascent+phagosomes+mature+to+become+phagolysosomes.">How nascent phagosomes mature to become phagolysosomes.</a> Trends Immunol. 33 (8), 397-405 (2012).</li><li>Nunes, P., Demaurex, N., Dinauer, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+NADPH+oxidase+and+associated+ion+fluxes+during+phagocytosis.">Regulation of the NADPH oxidase and associated ion fluxes during phagocytosis.</a> Traffic. 14 (11), 1118-1131 (2013).</li><li>Binder, B., Holzhutter, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hypothetical+model+of+cargo-selective+rab+recruitment+during+organelle+maturation.">A hypothetical model of cargo-selective rab recruitment during organelle maturation.</a> Cell Biochem Biophys. 63 (1), 59-71 (2012).</li><li>Hurtado-Lorenzo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=V-ATPase+interacts+with+ARNO+and+Arf6+in+early+endosomes+and+regulates+the+protein+degradative+pathway.">V-ATPase interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative pathway.</a> Nat Cell Biol. 8 (2), 124-136 (2006).</li><li>Weisz, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acidification+and+protein+traffic.">Acidification and protein traffic.</a> Int Rev Cytol. 226, 259-319 (2003).</li><li>Huynh, K. K., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+vacuolar+pH+and+its+modulation+by+some+microbial+species.">Regulation of vacuolar pH and its modulation by some microbial species.</a> Microbiol Mol Biol Rev. 71 (3), 452-462 (2007).</li><li>De Vito, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sodium/hydrogen+exchanger:+a+possible+mediator+of+immunity.">The sodium/hydrogen exchanger: a possible mediator of immunity.</a> Cell Immunol. 240 (2), 69-85 (2006).</li><li>Moreland, J. G., Davis, A. P., Bailey, G., Nauseef, W. M., Lamb, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anion+channels,+including+ClC-3,+are+required+for+normal+neutrophil+oxidative+function,+phagocytosis,+and+transendothelial+migration.">Anion channels, including ClC-3, are required for normal neutrophil oxidative function, phagocytosis, and transendothelial migration.</a> J Biol Chem. 281 (18), 12277-12288 (2006).</li><li>Winterbourn, C. C., Kettle, 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=Redox+reactions+and+microbial+killing+in+the+neutrophil+phagosome.">Redox reactions and microbial killing in the neutrophil phagosome.</a> Antioxid Redox Signal. 18 (6), 642-660 (2013).</li><li>Seredenina, T., Demaurex, N., Krause, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-Gated+Proton+Channels+as+Novel+Drug+Targets:+From+NADPH+Oxidase+Regulation+to+Sperm+Biology.">Voltage-Gated Proton Channels as Novel Drug Targets: From NADPH Oxidase Regulation to Sperm Biology.</a> Antioxid Redox Signal. , (2014).</li><li>Kotsias, F., Hoffmann, E., Amigorena, S., Savina, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+production+in+the+phagosome:+impact+on+antigen+presentation+in+dendritic+cells.">Reactive oxygen species production in the phagosome: impact on antigen presentation in dendritic cells.</a> Antioxid Redox Signal. 18 (6), 714-729 (2013).</li><li>Grimm, M. 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=Monocyte-+and+macrophage-targeted+NADPH+oxidase+mediates+antifungal+host+defense+and+regulation+of+acute+inflammation+in+mice.">Monocyte- and macrophage-targeted NADPH oxidase mediates antifungal host defense and regulation of acute inflammation in mice.</a> J Immunol. 190 (8), 4175-4184 (2013).</li><li>West, A. P., 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=TLR+signalling+augments+macrophage+bactericidal+activity+through+mitochondrial+ROS.">TLR signalling augments macrophage bactericidal activity through mitochondrial ROS.</a> Nature. 472 (7344), 476-480 (2011).</li><li>Bloomfield, G., Pears, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+signalling+required+for+multicellular+development+of+Dictyostelium.">Superoxide signalling required for multicellular development of Dictyostelium.</a> J Cell Sci.. 116 (Pt 16), 3387-3397 (2003).</li><li>Ramsey, I. S., Moran, M. M., Chong, J. A., Clapham, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+voltage-gated+proton-selective+channel+lacking+the+pore+domain.">A voltage-gated proton-selective channel lacking the pore domain.</a> Nature. 440 (7088), 1213-1216 (2006).</li><li>El Chemaly, A., Demaurex, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+Hv1+proton+channels+regulate+the+ionic+and+redox+homeostasis+of+phagosomes?.">Do Hv1 proton channels regulate the ionic and redox homeostasis of phagosomes?.</a> Mol Cell Endocrinol. 353 (1-2), 82-87 (2012).</li><li>El Chemaly, A., Nunes, P., Jimaja, W., Castelbou, C., Demaurex, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hv1+proton+channels+differentially+regulate+the+pH+of+neutrophil+and+macrophage+phagosomes+by+sustaining+the+production+of+phagosomal+ROS+that+inhibit+the+delivery+of+vacuolar+ATPases.">Hv1 proton channels differentially regulate the pH of neutrophil and macrophage phagosomes by sustaining the production of phagosomal ROS that inhibit the delivery of vacuolar ATPases.</a> J Leukoc Biol. , (2014).</li><li>Decoursey, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-gated+proton+channels.">Voltage-gated proton channels.</a> Compr Physiol. 2 (2), 1355-1385 (2012).</li><li>Ramsey, I. S., Ruchti, E., Kaczmarek, J. S., Clapham, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hv1+proton+channels+are+required+for+high-level+NADPH+oxidase-dependent+superoxide+production+during+the+phagocyte+respiratory+burst.">Hv1 proton channels are required for high-level NADPH oxidase-dependent superoxide production during the phagocyte respiratory burst.</a> Proc Natl Acad Sci U S A. 106 (18), 7642-7647 (2009).</li><li>Sturgill-Koszycki, 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=Lack+of+acidification+in+Mycobacterium+phagosomes+produced+by+exclusion+of+the+vesicular+proton-ATPase.">Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase.</a> Science. 263 (5147), 678-681 (1994).</li><li>Clemens, D. L., Lee, B. Y., Horwitz, 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=Virulent+and+avirulent+strains+of+Francisella+tularensis+prevent+acidification+and+maturation+of+their+phagosomes+and+escape+into+the+cytoplasm+in+human+macrophages.">Virulent and avirulent strains of Francisella tularensis prevent acidification and maturation of their phagosomes and escape into the cytoplasm in human macrophages.</a> Infect Immun. 72 (6), 3204-3217 (2004).</li><li>Alpuche-Aranda, C. M., Swanson, J. A., Loomis, W. P., Miller, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+typhimurium+activates+virulence+gene+transcription+within+acidified+macrophage+phagosomes.">Salmonella typhimurium activates virulence gene transcription within acidified macrophage phagosomes.</a> Proc Natl Acad Sci U S A. 89 (21), 10079-10083 (1992).</li><li>Lukacs, G. L., Rotstein, O. D., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+the+phagosomal+pH+in+macrophages.+In+situ+assessment+of+vacuolar+H(+)-ATPase+activity,+counterion+conductance,+and+H++'leak'.">Determinants of the phagosomal pH in macrophages. In situ assessment of vacuolar H(+)-ATPase activity, counterion conductance, and H+ 'leak'.</a> J Biol Chem. 266 (36), 24540-24548 (1991).</li><li>Watanabe, K., Kagaya, K., Yamada, T., Fukazawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+for+candidacidal+activity+in+macrophages+activated+by+recombinant+gamma+interferon.">Mechanism for candidacidal activity in macrophages activated by recombinant gamma interferon.</a> Infect Immun. 59 (2), 521-528 (1991).</li><li>Ip, W. 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=Phagocytosis+and+phagosome+acidification+are+required+for+pathogen+processing+and+MyD88-dependent+responses+to+Staphylococcus+aureus.">Phagocytosis and phagosome acidification are required for pathogen processing and MyD88-dependent responses to Staphylococcus aureus.</a> J Immunol. 184 (12), 7071-7081 (2010).</li><li>Clarke, M., Maddera, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocyte+meets+prey:+uptake,+internalization,+and+killing+of+bacteria+by+Dictyostelium+amoebae.">Phagocyte meets prey: uptake, internalization, and killing of bacteria by Dictyostelium amoebae.</a> Eur J Cell Biol. 85 (9-10), 1001-1010 (2006).</li><li>Jankowski, A., Scott, C. C., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+the+phagosomal+pH+in+neutrophils.">Determinants of the phagosomal pH in neutrophils.</a> J Biol Chem. 277 (8), 6059-6066 (2002).</li><li>Segal, A. W., Geisow, M., Garcia, R., Harper, A., Miller, 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+respiratory+burst+of+phagocytic+cells+is+associated+with+a+rise+in+vacuolar+pH.">The respiratory burst of phagocytic cells is associated with a rise in vacuolar pH.</a> Nature. 290 (5805), 406-409 (1981).</li><li>Savina, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NOX2+controls+phagosomal+pH+to+regulate+antigen+processing+during+crosspresentation+by+dendritic+cells.">NOX2 controls phagosomal pH to regulate antigen processing during crosspresentation by dendritic cells.</a> Cell. 126 (1), 205-218 (2006).</li><li>Rybicka, J. M., Balce, D. R., Chaudhuri, S., Allan, E. R., Yates, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagosomal+proteolysis+in+dendritic+cells+is+modulated+by+NADPH+oxidase+in+a+pH-independent+manner.">Phagosomal proteolysis in dendritic cells is modulated by NADPH oxidase in a pH-independent manner.</a> EMBO J. 31 (4), 932-944 (2012).</li><li>Mantegazza, A. 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=NADPH+oxidase+controls+phagosomal+pH+and+antigen+cross-presentation+in+human+dendritic+cells.">NADPH oxidase controls phagosomal pH and antigen cross-presentation in human dendritic cells.</a> Blood. 112 (12), 4712-4722 (2008).</li><li>Balce, D. R., Allan, E. R., McKenna, N., Yates, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gamma-Interferon-inducible+lysosomal+thiol+reductase+(GILT)+maintains+phagosomal+proteolysis+in+alternatively+activated+macrophages.">gamma-Interferon-inducible lysosomal thiol reductase (GILT) maintains phagosomal proteolysis in alternatively activated macrophages.</a> J Biol Chem. 289 (46), 31891-31904 (2014).</li><li>Sanjurjo, L., 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+scavenger+protein+apoptosis+inhibitor+of+macrophages+(AIM)+potentiates+the+antimicrobial+response+against+Mycobacterium+tuberculosis+by+enhancing+autophagy.">The scavenger protein apoptosis inhibitor of macrophages (AIM) potentiates the antimicrobial response against Mycobacterium tuberculosis by enhancing autophagy.</a> PLoS One. 8 (11), e79670 (2013).</li><li>Yuan, Z., Zhi, L., Li-Juan, Z., Hong-Tao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Utility+of+Chloroquine+in+Cancer+Therapy.">The Utility of Chloroquine in Cancer Therapy.</a> Curr Med Res Opin. , 1-12 (2015).</li><li>Ohkuma, S., Poole, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+probe+measurement+of+the+intralysosomal+pH+in+living+cells+and+the+perturbation+of+pH+by+various+agents.">Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents.</a> Proc Natl Acad Sci U S A. 75 (7), 3327-3331 (1978).</li><li>El Chemaly, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VSOP/Hv1+proton+channels+sustain+calcium+entry,+neutrophil+migration,+and+superoxide+production+by+limiting+cell+depolarization+and+acidification.">VSOP/Hv1 proton channels sustain calcium entry, neutrophil migration, and superoxide production by limiting cell depolarization and acidification.</a> J Exp Med. 207 (1), 129-139 (2010).</li><li>Levine, A. P., Duchen, M. R., Segal, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+HVCN1+channel+conducts+protons+into+the+phagocytic+vacuole+of+neutrophils+to+produce+a+physiologically+alkaline+pH.">The HVCN1 channel conducts protons into the phagocytic vacuole of neutrophils to produce a physiologically alkaline pH.</a> bioRxiv. , (2014).</li><li>Al-Fageeh, M. B., Smales, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+and+regulation+of+the+cellular+responses+to+cold+shock:+the+responses+in+yeast+and+mammalian+systems.">Control and regulation of the cellular responses to cold shock: the responses in yeast and mammalian systems.</a> Biochem J. 397 (2), 247-259 (2006).</li><li>Yui, N., 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=Basolateral+targeting+and+microtubule-dependent+transcytosis+of+the+aquaporin-2+water+channel.">Basolateral targeting and microtubule-dependent transcytosis of the aquaporin-2 water channel.</a> Am J Physiol Cell Physiol. 304 (1), C38-C48 (2013).</li><li>Griffiths, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+phagosome+individuality+and+membrane+signalling+networks.">On phagosome individuality and membrane signalling networks.</a> Trends Cell Biol. 14 (7), 343-351 (2004).</li><li>Poburko, D., Santo-Domingo, J., Demaurex, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+regulation+of+the+mitochondrial+proton+gradient+during+cytosolic+calcium+elevations.">Dynamic regulation of the mitochondrial proton gradient during cytosolic calcium elevations.</a> J Biol Chem. 286 (13), 11672-11684 (2011).</li></ol>

The authors have nothing to disclose.

虽然更多的时间比其他的方法，如光谱学和FACS，其采用使用pH敏感染料耦合到目标的一个类似的策略，但测量吞噬体的群体的平均pH值消耗，显微镜提供了几个优点。首先是，内部和外部的限制，但不内化，颗粒很容易被分辨，而无需添加其它化学品，如台盼蓝或抗体，以淬灭或标签外部粒子，分别。二是，继细胞进行实时让研究人员能观察吞噬过程中同步等迁移，传感和有约束力的粒子效果可?...

Phagocytosis, the process through which innate immune cells engulf large particles, evolved from the eating mechanism of single-celled organisms, and involves binding to a target, enveloping it with a membrane and pinching the membrane off to form a vacuole within the cytosol called a phagosome. While the phagosomal membrane is derived from the plasma membrane, active protein and lipid sorting, as well as fusion with endomembranes during phagosome formation, transform the phagosome into a distinct organelle within the cell with degradative properties that allow the killing, neutralization and breakdown of the ingested material1-3. This process, called phagosomal maturation, relies on the delivery of a host of proteolytic and microbicidal enzymes, including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase which transfers electrons into phagosomes producing the strong oxidant O2- and its derivative reactive oxygen species (ROS) 2,4. 
The luminal pH of phagosomes has a profound influence on several events required for phagosome function. First, pH influences trafficking of endomembranes in general, as pH-dependent conformational changes of transmembrane trafficking regulators alters the recruitment of trafficking determinants such as Arfs, Rabs and vesicular coat-proteins, which in turn define which vesicles may fuse with phagosomes 5-8. Second, the ionic composition of the phagosomal lumen is also transformed as phagosomes mature, and some ion transporters, such as the Na+/H+ exchanger or ClC family Cl-/H+ antiporters, which promote phagocytic function, rely on H+ translocation 9,10. Similarly, ROS production is intimately linked with phagosomal pH. ROS and its toxic oxidant byproducts have long been recognized as crucial for phagosomal killing in neutrophils 4,11,12, and have been shown to play critical roles in other phagocytes including macrophages, dendritic cells (DCs) and amoeba 13-16. The NADPH oxidase is an electrogenic enzyme that releases H+ in the cytosol as NADPH is consumed, and that requires the simultaneous transfer of H+ through companion HVCN1 channels alongside the transported electrons into the phagosomal lumen, in order to alleviate the massive depolarization that would otherwise lead to self-inhibition of the enzyme 17-21. Finally, several proteolytic enzymes have optimal activity at different pH, so time-dependent phagosomal pH changes can influence which enzymes are active and when. The importance of phagosomal pH is highlighted by organisms such as Mycobacterium tuberculosis, Franciscella tularensis and Salmonella typherium that subvert phagocytic killing at least in part by altering phagosomal pH 22-24.
In mammals the main phagocytes are neutrophils, macrophages and dendritic cells, and depending on cell type, time-dependent phagosomal pH changes can vary widely, and appear to play different roles. In macrophages a strong and rapid acidification mediated by the ATP-dependent proton pump vacuolar ATPase (V-ATPase) is one of the key factors mediating killing 25-27, resembling the mechanisms present in amoeba that use phagocytosis as an eating mechanism 28. In these cells activation of acidic proteases is thought to play a key role. In contrast, neutrophil killing relies more on ROS as well as HOCl produced by myeloperoxidase (MPO)11, and the pH remains neutral or alkaline during the first 30 min acidifying only later 29,30. Neutral pH has been suggested to favor the activity of oxidative proteases such as certain cathepsins. In DCs phagosomal pH is controversial, with some reporting acidification and others neutral or alkaline pH 31,32, but ROS and pH may profoundly influence the ability of these cells to present antigens to T cells, one of their main functions 33. 
Importantly, hormones, chemokines and cytokines may produce signaling events that induce maturation and changes in phagocyte behavior, and in turn influence phagosomal pH 34,35. Similarly, drugs, for example the antimalarial chloroquine, which is also considered for anti-cancer therapies 36, may affect phagosomal pH and therefore immune outcomes. Thus, a variety of cell biologists, immunologists, microbiologists and drug developers may be interested in measuring phagosomal pH when seeking to understand the mechanisms underlying the effects of a particular genetic disruption, bioactive compound or microorganism on innate and adaptive immune responses. 
Here we describe a method for measuring phagosomal pH in neutrophils that allowed us to show the importance of the HVCN1 channel in regulating neutrophil phagosomal pH 19. The method is based on the ratiometric property of fluorescein isothiocyanate (FITC) whose fluorescence emission at 535 nm is pH sensitive when excited at 490 nm but not 440 nm 37. When this dye is chemically coupled to a target, in this case zymosan, it can be followed using wide-field fluorescence microscopy, where cells are imaged as they phagocytose, and phagosomal pH changes are measured in real time as the phagosome matures. The actual pH is then gleaned by performing a calibration experiment where cells that have phagocytosed are exposed to solutions of different pH that contain the ionophores nigericin and monensin, that allow the rapid equilibration of the pH within phagosomes with the external solution. Ratio values are then compared to the known pH of solutions, a calibration curve is constructed by nonlinear regression and the resulting equation used to calculate pH from the ratio value.

<table>
	<tbody>
		<tr>
			<td>Zymosan A powder</td>
			<td>Sigma-Aldrich</td>
			<td>Z4250</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate</td>
			<td>Sigma-Aldrich</td>
			<td>F7250</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Anti-zymosan antibody (Zymosan A Bioparticles opsonizing reagent)</td>
			<td>Life Technologies</td>
			<td>Z2850</td>
			<td>Sigma-Aldrich O6637 is an equivalent product. Alternatively 25% serum can be used as an opsonizing reagent.</td>
		</tr>
		<tr>
			<td>4-Aminobenzoic hydrazide (4-ABH)</td>
			<td>Santa Cruz</td>
			<td>sc-204107</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Diphenyleneiodonium chloride (DPI)</td>
			<td>Sigma-Aldrich</td>
			<td>D2926</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Concanamycin A (ConcA)</td>
			<td>Sigma-Aldrich</td>
			<td>27689</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Nigericin</td>
			<td>Sigma-Aldrich</td>
			<td>N7143</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Monensin</td>
			<td>Enzo</td>
			<td>ALX-380-026-G001</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Life Technologies</td>
			<td>14200-075</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Hank&#39;s balance salt solution</td>
			<td>Life Technologies</td>
			<td>14025092</td>
			<td>Ringer&#39;s balanced salt solution or other clear physiological buffers may be substituted.</td>
		</tr>
		<tr>
			<td>Sodium carbonate (Na2CO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S7795</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>2-(N-Morpholino)ethanesulfonic acid (MES)</td>
			<td>Sigma-Aldrich</td>
			<td>M3671&#160;</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>N-Methyl-D-glucamine (NMDG)</td>
			<td>Sigma-Aldrich</td>
			<td>M2004&#160;</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid (EGTA)</td>
			<td>Sigma-Aldrich</td>
			<td>3777</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>&#160;Tris(hydroxymethyl)aminomethane (Tris)</td>
			<td>Sigma-Aldrich</td>
			<td>T1503&#160;</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Magnesium chloride (MgCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Absolute Ethanol (EtOH)</td>
			<td>Sigma-Aldrich</td>
			<td>2860</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Glass-bottom 35 mm petri dishes (Fluorodish)</td>
			<td>World Precision Instruments</td>
			<td>FD35-100</td>
			<td>Ibidi &#181;-clear dishes or coverslips with appropriate imaging chambers may be sustituted</td>
		</tr>
		<tr>
			<td>Sonicating water bath</td>
			<td>O. Kleiner AG</td>
			<td></td>
			<td>A sonicator may be used instead, various instrument providers exist</td>
		</tr>
		<tr>
			<td>Heamocytometer</td>
			<td>Marienfeld GmbH</td>
			<td></td>
			<td>Various instrument providers exist</td>
		</tr>
		<tr>
			<td>Widefield live imaging microscope</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td>Various instrument providers exist, but the microscope must be able to image 440/535 and 490/535 excitation/emission respective. Spinning disk confocal set-ups with brightfield capabilities may substituted, but zymosan tend to go out of focus more often.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Peristaltic pump (Dynamax RP-1)</td>
			<td>Rainin</td>
			<td></td>
			<td>Various instrument providers exist</td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Schott Gerate GmbH</td>
			<td></td>
			<td>Various instrument providers exist</td>
		</tr>
		<tr>
			<td>Manual Counter</td>
			<td>Milian SA</td>
			<td></td>
			<td>Various instrument providers exist</td>
		</tr>
	</tbody>
</table>

measuring phagosome ph by ratiometric fluorescence microscopy

吞噬作用是通过它先天免疫细胞吞噬细菌，细胞凋亡或其它外来粒子以杀死或中和摄入材料，或提供它作为抗原并引发适应性免疫反应的基本处理。吞噬体的pH值是一个重要的参数调节裂变或融合endomembranes和活化的蛋白水解酶，事件，允许吞噬液泡成熟为一个降解细胞器。此外，需要用于生产高水平的活性氧（ROS），它是必不可少的有效杀伤和信令到其他宿主组织的H +的易位。许多细胞内病原体通过限制吞噬体酸化，突出吞噬体生物pH值的重要性颠覆吞噬杀灭。在这里，我们描述了一个比例测量方法吞噬体pH值在使用异硫氰酸荧光素（FITC）标记的嗜中性粒细胞酵母多糖为吞噬TARGETS和活细胞成像。该测定法是基于FITC，，它是通过酸性pH时在490nm激发而不是当在440nm激发时，允许pH依赖比率的量化，而不是绝对的荧光，一单一染料的淬灭的荧光性质。还提供了用于进行原位染料校准和转换比实际的pH值的详细协议。单染料比例的方法通常被认为优于单波长或双染料伪比例协议，因为它们对扰动如漂白较不敏感，焦点改变，激光的变化，且不均匀标签，其中扭曲测量的信号。这个方法可以很容易地修改，以测量pH在其他吞噬细胞类型，和酵母聚糖可通过任何其它的含胺粒子取代，从惰性珠活的微生物。最后，该方法可以适于以利用其它荧光探针不同的pH值范围或其它phagosom敏感的人的活动，使之成为一个广义的协议吞噬小体的功能成像。

下面是有代表性的结果做了一个试验，其中初级小鼠嗜中性粒细胞从骨髓的野生型或分离的吞噬体的pH Hvcn1 - / - 小鼠进行比较。对于一个成功的实验，这是重要的定时影片的整个持续时间期间获得的视场范围内足够吞噬体，同时避免过多的吞噬体，其中图像分析过程中稍后将更加难以段。 图1示出了好的和坏的汇合的例子。对于时间相关和快照分析，外部酵母多...

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Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

由成比例的荧光显微镜测量吞噬体pH值

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

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Immunology and Infection

13.8K 次观看.  University of Geneva. Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy. 