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>

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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能会很容易在这里看出端倪，而这可能与人口为基础的方法忽略的多个方面。此外，吞噬作用的启动同步，经常通过放置细胞中进行，在4℃下，这可能会扰乱其他运输事件由于冷休克40,41，是无必要的时间为0 t的直接看出端倪。实际上，在执行现场显微镜时，这​​4℃同步技术不推荐，如果捕获的时间段接近吞噬作为温度偏移期间形成在盘底部的缩合可与目标浸油混合的引发在气候变暖的菜和镜组件之间的温差会导致重点转移。从这一事实的第三个优点茎吞噬体是在几个参数，包括本质上异构的，但不限于，pH值42，并在吞噬体的pH一定的影响可能更细微，改变吞噬体的pH而不是平均人口的分布，作为示于图3中 。这些观察可以得到更深机械见解吞噬体的pH的调节。 在本研究中，FITC标记被选为所选择的pH值敏感的染料，因为它是便宜，随处可见，重要的有6.5的pKa与动态范围涵盖pH为3至9日在其免费的形式37，其匹配的中性至微碱性的原先预期的pH值范围内，根据以往的研究29,30。最近采用所谓SNARF不同pH探头，其具有7.5的更基本的pKa值，意外地估计的pH值范围为野生型和Hvcn1 1-2个pH单位更碱性的一项研究- / -嗜中性粒细胞吞噬体39。因此，pH传感器的选择是考虑根据应用的一个重要参数。原则上，在这里提出的方法可以容易地适合于采用其它pH值敏感的染料，如SNARF和2&#39;，7&#39;-二（2-羧乙基）-5-（和-6） - 羧基（BCECF），其琥珀酰亚胺基酯衍生物容易地连接到含胺颗粒，如酵母聚糖。事实上，传感器不必限制于荧光染料，如ratiometRIC pH敏感的蛋白质，这样的SypHer 43，可以被转导的和由微生物表达的。几个非比例的替代也是存在的，特别是红色版本染料pHrodo，其可在与绿色荧光蛋白（GFP）一起使用的-tagged蛋白质或pH敏感蛋白pHluorin。由于潜在的大的效果的严酷环境intraphagosomal可以对靶向于其中染料和蛋白质然而，这些探针应在最低结合pH值不敏感的探针在导出伪比率要采用。例举在图5中，校准实验可以公开的染料的损害作为减少或扭曲的动态范围的影响。在实践笔记，标定实验并不总是完美的，可能是不可行的每一个实验后执行。因此，比值可能偶尔掉出校准的范围和转换​​时产生不可能值至pH。它可以appropr黄昏时分要么表达这些超出量程值"大于/小于或等于"最大和最小刻度的pH值，或表达的所有值比和估计pH范围的平均比率表示。 所强调的DPI和孔卡控制，ROS的产生和V-ATP酶的活性是决定吞噬体pH值的主要因素。事实上，在许多情况下，改变ROS产生的水平可能在背后吞噬体pH值的差异，特别是在中性粒细胞和吞噬体ROS产生的测量和pH值往往齐头并进。谨慎的一个字是，两种药物借给洞察它们抑制但不向细胞内的位置中的酶的活性。两种酶是多亚基复合物，其各个组成部分必须流量吞噬体4，和缺乏对吞噬体的活性可能源于贩卖或组装缺陷而不是直接影响。

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>Toxic, use gloves, various providers exist</td></tr><tr><td>Hank&#039;s balance salt solution</td><td>Life Technologies</td><td>14025092</td><td>Ringer&#039;s balanced salt solution or other clear physiological buffers may be substituted.</td></tr><tr><td>Sodium carbonate (Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;)</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&amp;nbsp;</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&amp;nbsp;</td><td>Various providers exist</td></tr><tr><td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&amp;prime;,N&amp;prime;-tetraacetic acid (EGTA)</td><td>Sigma-Aldrich</td><td>3777</td><td>Various providers exist</td></tr><tr><td>Tris(hydroxymethyl)aminomethane (Tris)</td><td>Sigma-Aldrich</td><td>T1503&amp;nbsp;</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 (MgCl&lt;sub&gt;2&lt;/sub&gt;)</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 &amp;micro;-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.&amp;nbsp;&amp;nbsp;</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，外部酵母多糖颗粒必须从吞噬体区分开来，并从分析中排除。 图2示出了明场图象如何能帮助从吞噬体区分外部酵母聚糖颗粒是有用的。 图3示出了典型的吞噬体的例子相比Hvcn1 pH值时，课程在野生型- / - </EM&gt;中性粒细胞，年均pH值，并吞噬体pH值群体的直方图。这些结果凸显的情况下，平均人口pH值不变，而pH值的分布强烈不同。良好控制是始终重要的。 图4示出增加的NADPH氧化酶抑制剂的DPI，这导致了快速酸化，随后加入在V-ATP酶抑制剂孔卡，其反转酸化的效果。这样的实验可以是临界证明pH敏感探针吞噬体中工作，他们应该。典型的校准时间过程示于图5中，用矩形表示用于构建校准曲线比的值的范围。图5另外示出了如何染料通过MPO活性氯化可以影响的FITC的intraphagosomal pH的响应，突出的事实该phagoso内比预期的探头可以有不同的反应<html>发作环境和原位校准的重要性。 <img alt="图1" src="/files/ftp_upload/53402/53402fig1.jpg" /> 图1.适当汇合和目标:细胞比例左侧面板显示了一个例子，其中细胞汇合过于稀疏和目标:细胞比例过低，减少一个吞噬事件将发生在视场的概率。中间面板显示了一个例子，其中细胞汇合过高和细胞拥挤。在这些条件下，细胞可能没有足够的空间来移动，并找到他们的目标，或者可以抓取更多困难以后在彼此之上制作分析。右图显示一个理想的细胞汇合和初始目标:细胞比例，其中许多小区和目标可被看见，但仍然可以清楚地从每个彼此区分。 FITC-酵母聚糖显示为绿色和比例尺条为10微米。<html> /ftp_upload/53402/53402fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/53402/53402fig2.jpg" /> 图2.区分吞噬体和uningested酵母多糖，左边的面板中显示了合并后的亮场图像和四百四十分之四百九十〇 FITC-酵母多糖的比例，而右边面板显示了亮场图像孤单。吞噬小体（小绿箭头）可以从外部颗粒区别开来，要么不共定位与细胞（短的红色箭头），或者其荧光不与黑暗的中心（长的红色箭头），匹配由亮环围绕，特征在亮场图像（绿色箭头）吞噬体。该比例尺代表10微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53402/53402fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> ENT"FO:保持together.within页="1"&gt; <img alt="图3" src="/files/ftp_upload/53402/53402fig3.jpg" /> 图3.时间场，野生型和Hvcn1的平均pH值和比直方图- / - 。嗜中性粒细胞吞噬体（A）的吞噬体pH值的典型时间进程显示，而在野生型嗜中性粒细胞（蓝色）的pH为中性（虚线）或以下摄取，Hvcn1的吞噬体微碱性（实线）在早期时间点- / -嗜中性粒细胞（红色）可以碱化（实线）或酸化（虚线）。 （B）中编译的快照取30-35分钟后加入目标允许平均人口吞噬体的pH可以从大量吞噬体（野生型的计算:N = 5/1898年/ 383和Hvcn1 - / - :N = 6 /四百五十一分之一千四百三十三实验/吞噬体/细胞，酒吧都在平均值±SEM，NS不显著）。 FITC-zymosa（三）直方图N比揭示野生型和Hvcn1之间的差异在吞噬体pH值- / -嗜中性粒细胞。 （这个数字已经被修改[19]，许可。） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53402/53402fig3large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53402/53402fig4.jpg" /> 图上吞噬体的pH 4.影响NADPH氧化酶抑制剂的DPI和V-ATP酶抑制剂孔卡。预培养在10μM的DPI导致吞噬体的迅速酸化摄取后不久，这是通过加入100nM的孔卡（箭头）逆转。 （这个数字已经被修改[19]，许可。） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53402/53402fig4large.jpg" target="_blank">点击此处查看该图的放大版本。</a> 图5. pH校准时间进程和FITC-酵母多糖的校准MPO的抑制效果。（A）intraphagosomal FITC-酵母多糖的典型PH校准曲线。红色的框表示用于构建校准曲线比的值的范围。所使用的解决方案，并在该溶液被改变的次数的实际的pH值由箭头指示。该曲线表示的视图中的单个场在10吞噬体的平均值。 MPO的（B）中抑制与非特异性抑制剂叠氮化钠（5mM的，MPO INH）增加的intraphagosomal FITC的酵母聚糖pH依赖性反应的动态范围。点是平均值±SEM（这个数字已经被修改[19]，许可。） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53402/53402fig5large.jpg" target="_blank">点击此处查看一拉</a>rger版本这个数字。 <table><tbody><tr><td> 1.1 </td><td> （2个）基地</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>缓冲区</td><td>股票</td><td>决赛（1X） </td><td> 500毫升（2×） </td><td></td><td></td></tr><tr><td></td><td>氯化钾</td><td> 1M的</td><td> 140毫米</td><td> 140毫升</td><td></td><td></td></tr><tr><td></td><td> 氯化镁 </td><td> 1M的</td><td> 1毫米</td><td> 1毫升</td><td></td><td></td></tr><tr><td></td><td> EGTA </td><td> 1M的</td><td> 0.2毫米</td><td> 0.2毫升</td><td></td><td></td></tr><tr><td></td><td>氯化钠</td><td> 1M的</td><td> 20毫米</td><td> 20毫升</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 1.2 </td><td>缓冲区</td><td>股票</td><td>最终（1X） </td><td>目的</td><td></td><td></td></tr><tr><td></td><td> MES </td><td> 1M的</td><td> 20毫米</td><td> pH值5.5-6.5 </td><td></td><td></td></tr><tr><td></td><td> HEPES </td><td> 1M的</td><td> 20毫米</td><td> pH值7.0-7.5 </td><td></td><td></td></tr><tr><td></td><td>三</td><td> 1M的</td><td> 20毫米</td><td> pH值5.5-6.7 </td><td></td><td></td></tr><tr><td></td><td> NMDG </td><td> 1M的</td><td> 20毫米</td><td> pH值5.5-6.8 </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 1.3 </td><td>离子载体</td><td>库存（100％乙醇） </td><td>决赛（1X） </td><td></td><td></td><td></td></tr><tr><td></td><td>尼日利亚</td><td> 5毫克/毫升</td><td> 5微克/毫升</td><td></td><td></td><td></td></tr><tr><td></td><td>莫能菌素</td><td> 50毫米</td><td> 5μM </td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 1.4 </td><td>对于50毫升</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td> pH值</td><td> 2个基地</td><td>缓冲区类型</td><td>缓冲卷。 </td><td>尼日利亚</td><td>莫能菌素</td></tr><tr><td></td><td> 5 </td><td> 25毫升</td><td> MES </td><td> 1毫升</td><td> 50微升</td><td> 5微升</td></tr><tr><td></td><td> 5.5 </td><td> 25毫升</td><td> MES </td><td> 1毫升</td><td> 50微升</td><td> 5微升</td></tr><tr><td></td><td> 6 </td><td> 25毫升</td><td> MES </td><td> 1毫升</td><td> 50微升</td><td> 5微升</td></tr><tr><td></td><td> 6.5 </td><td&gt;25毫升</td><td> MES </td><td> 1毫升</td><td> 50微升</td><td> 5微升</td></tr><tr><td></td><td> 7 </td><td> 25毫升</td><td> HEPES </td><td> 1毫升</td><td> 50微升</td><td> 5微升</td></tr><tr><td></td><td> 7.5 </td><td> 25毫升</td><td> HEPES </td><td> 1毫升</td><td> 50微升</td><td> 5微升</td></tr><tr><td></td><td> 8 </td><td> 25毫升</td><td>三</td><td> 1毫升</td><td> 50微升</td><td> 5微升</td></tr><tr><td></td><td> 8.5 </td><td> 25毫升</td><td>三</td><td> 1毫升</td><td> 50微升</td><td> 5微升</td></tr><tr><td></td><td> 9 </td><td> 25毫升</td><td>三</td><td> 1毫升</td><td> 50微升</td><td> 5微升</td></tr><tr><td></td><td> 9.5 </td><td> 25毫升</td><td> NMDG </td><td> 1毫升</td><td> 50微升</td><td> 5微升</td></tr><tr><td></td><td colspan="2">调节pH值KOH或HCl </td><td></td><td></td><td></td><td></td></tr></tbody></table> 表1.配方制备的校准解决方案。 1.1）配方制备的2×基液500毫升1.2）配方制备使用了不同的缓冲液根据校准溶液的pH值1.3）配方用于制备校准溶液中使用的离子载体。1.4）配方制备50毫升用在1.1-1.3中描述的碱，缓冲液和离子载体每个校准溶液。

Watch this Scientific Journal Video about Measuring Phagosome pH by Ratiometric Fluorescence Microscopy at JoVE.com

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 ...

measuring-phagosome-ph-by-ratiometric-fluorescence-microscopy

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

13.9K 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. 

视频: Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

在现场研究巨噬细胞吞噬溶酶体生源

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

科研

免疫与感染

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

高通量荧光技术巨噬细胞吞噬功能和肌动蛋白聚合的评估

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

"吞噬体封闭试验"可视化吞噬体形成的三个维度利用全内反射荧光显微镜（TIRFM）

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement.

This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.

成像中性粒细胞吞噬体和细胞质使用比例测量pH指示剂

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

单细胞荧光显微镜定量 Efferocytosis 的研究

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Measuring the pH, Redox Chemistries, and Degradative Capacity of Macropinosomes using Dual-Fluorophore Ratiometric Microscopy

In recent years, the field of macropinocytosis has grown rapidly. Macropinocytosis has emerged as a central mechanism by which innate immune cells maintain organismal homeostasis and immunity. Simultaneously, and in contrast to its homeostatic role, it can also drive various pathologies, including cancer and viral infections. Unlike other modes of endocytosis, the tools developed for studying the maturation of macropinosomes remain underdeveloped. Here the protocol describes newly developed tools for studying the redox environment within the lumen of early and maturing macropinosomes. Methodologies for using ratiometric fluorescence microscopy in assessing the pH, production of reactive oxygen species, and the degradative capacity within the lumen of individual macropinosomes in live cells are described. Single organelle measurements offer the advantage of revealing spatiotemporal heterogeneity, which is often lost with population-based approaches. Emphasis is placed on the basic principles of dual fluorophore ratiometric microscopy, including probe selection, instrumentation, calibration, and single-cell versus population-based methods.

We describe protocols for measuring pH, oxidative events, and protein digestion in individual macropinosomes in live cells. An emphasis is placed on dual-fluorophore ratiometric microscopy and the advantages it offers over population-based techniques.

使用双氟比度显微镜测量巨无素体的pH值、重度化学和降解能力

In recent years, the field of macropinocytosis has grown rapidly. Macropinocytosis has emerged as a central mechanism by which innate immune cells ...

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases2. Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells3. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.

Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.

同时进行pH测量的内吞和胞车厢中使用共焦显微镜活细胞

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial ...

生物学

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro&#160;microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an &#34;eat-me&#34; signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging.

iPSC-巨噬细胞死亡神经母细胞的体外定量成像检测

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. ...

神经科学

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

利用pHluorin标记的受体监视亚细胞定位和贩运

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

TIRF Microscopy-Based Visualization of Phagosome Formation and Closure

For non-covalent fixation of the SRBCs, pour 2 milliliters of the IgG-opsonized cells into each of the poly-lysine-coated dishes, and centrifuge the samples in a swinging rotor centrifuge. Discard the supernatants, and wash the particles one time with 2 milliliters of PBS plus 10% BSA.
Then, incubate the particles with 2 milliliters of fresh PBS plus 10% BSA for 30 minutes at room temperature, followed by three washes with 2 milliliters of PBS. After the last wash, replace the PBS with 2 milliliters of 37 degrees Celsius serum-free microscopy medium.
Place an SRBC-coded dish onto the microscope stage. Then, scrape the transfected cells of interest from the bottom of the culture dish and pipette the cells a few times to achieve a single-cell suspension. Add the transfected cells to the SRBC-coated dish.
Start the live acquisition software. Find a cell that expresses the fluorescently tagged proteins and adjust the position of the dish so that a cell of interest is in the middle of the field. Acquire 500 images at different angles from 0 to 5 degrees at 0.01-degree increments at one excitation wavelength.
To determine the critical angle for the incident light to be totally reflected at the glass-medium interface and generate an evanescent wave, open the image sequence in the appropriate imaging software. Select a region of interest in the cell with uniform fluorescence.
Then, under the Image tab, select Stacks and plot Z-axis profile. To plot the Z-axis profile mean fluorescence intensity measured in the region of interest with the function of the angles on the X-axis. Any angle value on the x-axis superior to the critical angle can then be used during the microscopy session to obtain a TIRF signal.