这项工作是由好心威康信托，生物技术和生物科学研究理事会和欧文·乔夫纪念奖学金资助。

伦理声明:所有的动物工作与许可和英国内政部的批准下进行。在这项研究中人类参与的人类研究伦理被批准为联合UCL / UCLH委员会。所有参加者签署知情同意书。 1. 白色念珠菌的制备<ol><li>成长念珠菌盘显示（参见材料列表）上的YPD琼脂平板上按照制造商的说明。挑选一个菌落，并将其添加到15毫升YPD肉汤中。孵育它在振荡培养箱中于30℃和200rpm下，直到液是混浊（通常约2天，或至大约在1×10 9 / mL的浓度）。 </li><li>降速念珠菌介质和在50mL磷酸盐缓冲盐水（PBS）的重悬它。离心机中以3000×g离心10分钟。重复此步骤两次。 </li><li>放置含50毫升PBS / 念珠菌介质的管在预热至60℃的水浴中，以使整个管为submerged 1个小时。 <ol><li>为了确认念珠菌是热灭活的，条纹的样品到YPD琼脂平板上，并在30℃下孵育过夜。调整热灭活（HK） 念珠菌浓度为5-9×10 8个 / ml，取决于念珠菌生长，并且它在一个-20存储在1mL等分试样℃冷冻机中。 注意:使用自动细胞计数器进行该协议中的所有细胞计数。 </li></ol></li></ol> 2. S-1耦合到热杀死（HK） 念珠菌 <ol><li>通过在100μL高档二甲基亚砜（DMSO）稀释制备羧基-S-1琥珀酰亚胺酯（50微克）的等分试样。涡以及混合。 </li><li>在一个15毫升管中准备在0.1M碳酸氢钠（pH为8.3）1毫升1×10 8 HK 念珠菌 。 </li><li>添加100μL羧基-S-1在时刻到HK 念珠菌 ，同时在涡旋混合以大约2000rpm的转速（中到高速）一滴。包装铝FOI升管周围，并将其放置在滚筒上在室温下1个小时。 </li><li>通过以2250×g下，每次10分钟洗HK Candida- S-1（HKC-S-1）的三倍。对于前两个洗涤，重新悬浮在15毫升的0.1M碳酸氢钠（pH值8.3）的沉淀。在第三次洗涤后，重悬它在1mL平衡盐溶液（BSS）的缓冲液（ 见表1）。 </li><li>转移HKC-S-1悬浮液为在100μL等分试样管，并将它们存储在-20℃。 注:具有低表面结合材料，如果可能的话，使用如管HKC-S-1颗粒可能会粘到正常离心管的壁上。 </li><li>调理HKC-S-1 <ol><li>对于人嗜中性粒细胞，人IgG的血清加入100微升至100微升的解冻HKC-S-1。 </li><li>对于小鼠嗜中性粒细胞，加入正常小鼠血清的50μL和小鼠念珠菌免疫血清的50μL5至100μL（从与HK 念珠菌注射C57B6小鼠中产生）的HKC-S-1。 </li><li>混合它在热摇床在37℃和1,100rpm下60-90分钟，然后在4℃下辊2小时。 </li><li>通过以17200×g离心洗BSS缓冲液三次，每次1分钟。重悬在100μLBSS缓冲液中。 </li></ol></li></ol> 3.中性粒细胞的分离<ol><li>对于人外周血嗜中性粒细胞，从健康供体取15毫升的血液通过静脉穿刺，将其放入含有90μL的1000 IU / mL肝素钠溶液（60微升肝素/ 10毫升的血液）在20mL注射器中。 </li><li>使用移液管尖端，注入1.5毫升的10％的葡聚糖溶液到注射器。轻轻翻转的3倍，让它在板凳上直立。 </li><li>等待30-60分钟，直到血液被对半大致分裂，具有顶部血沉棕黄层是稻草色和包含红细胞的底层。 </li><li>小心通过针推出前层或用移液管尖端将其删除。把它放在我呐15mL的管中。避免服用了红层。使用5毫升吸管，加入3-4毫升的密度梯度介质的（见材料列表）的血沉棕黄层下的管，以获得两个不同的层的底部。离心机中以913×g离心10分钟。 注意:如果使用小鼠嗜中性粒细胞（参见附图17为嗜中性粒细胞的小鼠隔离），使用已被从骨髓冲洗代替细胞悬浮液。 </li><li>倒掉上清，留下红色沉淀后面。轻轻涡旋打扰颗粒。添加7毫升蒸馏水，高压灭菌H 2 O与沉淀和反转20秒，以重悬沉淀。加入7毫升2X生理盐水和颠倒几次混合，裂解剩余的红血细胞。 </li><li>离心机中以300×g离心5分钟。倒掉上清，重悬在BSS缓冲沉淀到大约4×10 6 /毫升。 注:对于细胞的最佳显微镜，保持1.5-6×10 6之间的细胞悬浮液的浓度</SUP&gt; /毫升。 </li></ol> 4.幻灯片的制备<ol><li>预治疗的8孔显微镜板（参见材料列表）与多聚-L-赖氨酸的200μL（0.01％溶液）在各孔中，在室温下40-60分钟。 </li><li>除去聚-L-赖氨酸（可重复使用），并用蒸馏水H 2 O的200μL洗涤孔两次</li><li>添加在步骤3.6制得到各孔的细胞悬浮液的200μL。在室温下孵育30-60分钟。 </li><li>通过添加100μL高档DMSO的一个反应管中准备的5-（和-6） - 羧基S-1乙酰氧基甲基（S-1-AM）酯（50微克）的等分试样。涡旋混合。当不使用时，储存在-20℃。 </li><li>在小管中，加入1.7 mL的BSS缓冲液和S-1-AM的20μL。涡旋混合。 </li><li>用BSS缓冲液200μL洗涤各孔两次，然后替换为S-1-AM溶液的缓冲液中。注意不要轻轻吹打扰乱连接到井底细胞的单层顺着孔壁。在室温下孵育至少25分钟。 </li><li>用BSS缓冲液200μL洗涤各孔两次。为了测试抑制剂，弥补在BSS缓冲适当药物的"主混合物"。在抑制剂溶液洗涤各孔两次。 注意:确保在控制使用的药物载体（ 例如，DMSO或乙醇）的相同量以及用于该抑制剂。如果在测试的氯化锌的效果，使用缺乏KH 2 PO 4缓冲液BSS（在HEPES缓冲液可以单独溶液），作为锌的磷酸盐沉淀。 </li><li>声处理调理HKC-S-1（第6.2节）中在5微米振幅大约3秒。加入10微升至每孔。孵育在37℃下将板15-20分钟，以允许细胞吞噬，使细胞准备要被成像为吞噬作用的快照。 </li></ol> 5.共聚焦显微镜<ol><li>使用共焦显微镜，调节激光波长，使得第在所述细胞是在555nm处激发和发射是由两个通道或检测器检测:560-600纳米和600纳米。 注意:特定显微镜参数将为每个显微镜不同，并且需要由研究人员进行优化。 </li><li>查看使用63X镜头油细胞。使用上连续设置一个瓦片扫描图像来查看中心瓦片。使用荧光强度和检测器通道的增益，调整焦距和激光的强度和两个信道的增益以优化图像。 </li><li>使用的设置，以查看两个通道分割图像;检查有在细胞质或空泡没有荧光强度的饱和。饱和度显示为红色圆点。降低激光的强度，以便有红点的最少数量，但这些细胞和吞噬足够明亮清楚地看到。 </li><li>满意后，点击"开始实验。" A 9瓦片扫描图像被生成。将图像保存为一个复杂的文件建议包含两个信道（未JPEG或TIFF）的一个合成图像的软件。 </li></ol> 6.标定实验<ol><li> 构造具有单独念珠菌 pH值标准曲线。 <ol><li>准备从pH为3.0缓冲器至13.0，按表1中 。 </li><li>采取HKC-S-1中的一个等分试样（100微升）。旋下来在17200×g的1分钟。重悬它在所分配的缓冲器500μL。 </li><li>从最低的pH范围内开始，悬浮液添加到一个预处理阱（步骤4.2）。放置在共聚焦显微镜载玻片上设置为37℃的加热阶段。 </li><li>记录荧光每分钟10分钟。重复每个缓冲区。 </li></ol></li><li> 皂素标准曲线。 <ol><li>隔离1×10 7人中性粒细胞5和重悬在1mL的最低pH值缓冲液中。 </li><li>按照第4，用于测量页记载的方法hagosomal pH值在中性粒细胞。 </li><li>与念珠菌悬浮液20分钟温育后，0.3％皂苷加入（10％储备的15μL到一个预处理阱（来自步骤4.2））的培养皿中，然后在37℃下孵育20分钟。拍摄图像每分钟10分钟。重复每个缓冲区。 </li></ol></li><li> 使用高K + /尼日利亚菌素技术 5，18，19 胞质pH标准曲线 。 <ol><li>弥补在表1中所描述的，从pH4.0至13.0的缓冲液。 </li><li>按照步骤4.1-4.7，只留下嗜中性粒细胞在预处理的孔 - 每个孔包含不同的缓冲器。滑动添加到37℃下加热阶段并记录每分钟10分钟的荧光。 </li><li>注意:可能需要一些时间对胞质pH至与外部溶液稳定，但在该点之后，在S-1的比例值变为不变。在每个实验中测定细胞外HKC-S-1可以作为控制来检查添加任何抑制剂不与S-1发射的荧光干扰或影响该缓冲区的pH值。 </li></ol></li></ol> 7.图像分析注:在这里，提供了使用免费软件ImageJ的图像分析（荧光定量）的说明。建议ImageJ的使用。 <ol><li>下载并安装ImageJ的版本&gt; 1.46r按照开发者的指令（https://imagej.nih.gov/ij/）。安装附有此协议称为补充代码文件"吞噬体测量"。 </li><li>打开图像J和加载用于分析选择到工具栏上的图像文件。要结合这两个通道，使用图像&gt;彩色&gt;制造复合。 </li><li>右键单击，选择用于存储结果的文件。 </li><li>点击行工具工具栏上，然后双击来增加宽度＆＃34; 2"。 </li><li>绘制跨越吞噬体的宽度的线，然后单击右键"测量的pH"。两个通道的平均强度被获取时，以及它们的比率通过ImageJ中的代码文件中自动计算出来。 </li><li>在完成测量后，用鼠标右键单击，选择"保存文件"。 </li><li>为了测量吞噬面积，使用第四个图标沿工具栏绘制手绘周围的区域。右键单击选择"测量面积。" 注:结果保存在选定的目录中生成一个日志文件。结果可以使用电子表格，R，或等价的软件进行处理。至pH荧光比之间的关系是S形近似，并且通过使用ImageJ分析生成的比率值可以使用广义后勤或S型函数或由直链的或三次样条插值被转换为pH值。 </li><li>为了测量细胞质，得出整个细胞质的线，然后右键单击"测量pH值"。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

一旦适当的试剂，显微镜设置和校准实验设置，这种方法是相对简单的执行。的关键步骤包括:与S-1标记的念珠菌 ，以确保不存在染料的超载，校准和分析图像。 S-1是适合于碱性pH环境的试剂，它是嗜中性粒细胞21特别重要，但限制在某些细胞类型及其用途。有关详细的酸性环境，如巨噬细胞吞噬体，SNARF-4，或S-4，是更合适的，因为其下的pKa 22。此外，对于更精确的读数细胞质，最好是使用S-4，作为用于S-1的标准曲线示出了荧光比开始低于pH 6高原（ 图3）。其他染料，如2&#39; ，7&#39;-二（2-羧乙基）-5-（和-6） - 羧基荧光素（BCECF）或红色的pHrodo也可能更适合在CONT分机，预计将是酸性的。然而，胞浆染色仍是必要的正确识别含念珠菌的吞噬的。 一个吞噬体pH指示剂的一个重要特征是，它是不可逆的通过反应性吞噬体环境改变。 S-1似乎是中性粒细胞性环境。这是通过Levine 等人示出。 5（参见参考文献5补充视频4），其表明由Hvcn1吞噬和S-1标记的念珠菌粒子的随后的释放- / -嗜中性粒细胞。当吞噬，颗粒从黄色/橙色到红色（中性至碱性pH下）转动时，但是当粒子通过嗜中性粒细胞释放，它返回到原来的颜色。 重要的是要提一些使用S-1相关的限制是非常重要的。这染料具有两个发射光谱允许比率MEAS的事实urement是一个优势，但需要专门的设备来获取图像;用于实验的显微镜必须能够记录同时或与非显着的时间延迟的两个图像。作者假设，研究人员在尝试此协议利用共聚焦显微镜有经验，或访问训练有素的专业人员。我们无法列出所有的特定显微镜参数，因为它们会为每个显微镜不同，需要由研究人员进行优化。缀合到S-1的乙酰氧基甲基酯，其允许染料扩散进入细胞的细胞质是通过在细胞的细胞质中的非特异性酯酶降解以形成荧光分子。酯酶，如碱性磷酸酶，存在人血清和胎牛血清，其用于补充细胞培养基英寸因此，在其中所述细胞加载S-1-AM（第4.5节）所述介质必须不含有血清。如果使用需要更多的营养RIC细胞，这可能具有挑战性H培养基，以维持它们比贯穿本协议中使用的平衡盐溶液。类似地，其他荧光培养基组分，如酚红，可与S-1的测量干扰。 图3中的误差棒表明，有在各pH的比率测量的一些变化。合适数目的每个实验的重复（至少n = 3）并且在每个单次实验中作为许多单个的测量需要克服间液泡的变化。因此建议测量至少100个吞噬体对于每种条件并显示到仅包含一个念珠菌尽可能多的吞噬体区域的pH。用于定量应该是那些已完全吞噬念珠菌粒子（ 即，那些完全被细胞质包围）将待测量的吞噬体。为了减轻防止意外偏差在用于定量细胞/吞噬体的选择，所有的分析应而进行盲目的实验条件。 在这里，我们的全血接着等离子体层的离心分离通过密度梯度葡聚糖沉降描述嗜中性粒细胞的分离。我们使用这个技术，因为它快速和有效地产生嗜中性粒细胞的纯（&gt; 95％）的人口，虽然也有可用的其它方法，例如通过其他密度梯度公式或使用专门的试剂盒与抗体或磁中性粒细胞的阴性选择全血离心珠。然而，后者可以是谁经常嗜中性粒细胞中分离大多数组昂贵。另外，我们使用抗凝血剂肝素的血液收集管，而其它研究人员可能更习惯于使用乙二胺四乙酸（EDTA）或酸式柠檬酸盐钠（ACD）。由于有许多不同的方法可供选择，它是由研究者的个人喜好。 此外，分离和操纵嗜中性粒细胞时它们应与一些小心处理以避免过度活化。预防措施包括:使用只塑料器皿，没有玻璃;过滤灭菌的所有缓冲区，以除去任何污染的内毒素;当纺纱嗜中性粒细胞确保离心机是很好的平衡，避免过度振动;限制尽可能时间中性粒细胞保留在离心后的颗粒;不保持在超过5×10 6个 / mL的溶液的嗜中性粒细胞;和隔离后尽快进行实验。 可以适于该方法通过使用显微镜加热的阶段设定为37℃和从相同位置拍摄快照每隔30-60秒至测量随时间的pH和吞噬体面积的变化，作为用于校准的步骤进行说明。它也可以在理论上适合于高通量的实验，例如在96孔板中，并进行流式细胞术实验，其中S-1可以被用作pH指示剂23。然而，在这些设置，强调个人的细胞活性是通过在细胞群更具全球性的影响所取代。 这种方法旨在提供一个比较简单的实验装置在个别研究人员可以适应，以满足他们感兴趣的领域。研究人员可能希望探索嗜中性粒细胞吞噬体的pH值和面积，同时还测量其它离子的移动，例如，细胞内钙浓度。有几种荧光钙指标容易地用于共焦显微镜，如印1，其还具有在400和475nm处24双发射光谱。这些发射波长不与S-1的发射光谱重叠，但激发波长是在光谱，其可以破坏细胞的紫外线（UV）端，和一个紫外激光是不是在所有显微镜普遍。不同指标的全面审查测量细胞内钙通量由高桥等人所覆盖。 25和希尔森等。 26。 总之，存在可使用不同的荧光染料如生菜等来测量pH为吞噬体的其它方法。已经证明13以及其它基团27,28。其他研究人员也使用S-1来测量pH为胞质29或吞噬体的pH 14。然而，该协议是在同时测量两个胞质溶胶和吞噬体的pH值，这提供了机会来观察吞噬体的横截面面积的变化和野生型和Hvcn1区分独特- / -小鼠嗜中性粒细胞，和具有和不具有人嗜中性白细胞工作NADPH氧化酶。

中性粒细胞是人体内最丰富的先天免疫细胞。它的主要功能包括巡逻血流和吞噬并消化，它可能在被称为吞噬1,2的过程遇到外来颗粒。这些颗粒中降解称为吞噬细胞内舱。中性粒细胞NADPH氧化酶的亚型NOX2，活化引发生物化学反应的级联，在病原体死亡高潮。 NOX2蛋白质亚基进行到形成在吞噬体膜3的电子传递链复合体。一旦被激活，它从NADPH穿过膜传输电子到分子氧吞噬体内的，从而产生超氧阴离子和进一步的活性​​氧物种。这被称为呼吸爆发，并且它被认为是用于有效微生物杀伤和消化2必不可少</suP&gt;。然而，负电荷穿过膜这个独特的运动很快就会失活NOX2，如果它不是由正电荷移动的和/或负电荷移出吞噬的补偿。已经公认的是大多数在中性粒细胞电荷补偿由质子通道HVCN1 4,5进行。这个信道允许质子向下从细胞质到吞噬体它们的电化学梯度的被动运动。质子浓度通过pH反射，所以对于氧化酶活性的给定电平，测量pH在吞噬体可以提供在电荷补偿质子和非质子途径的相对参与信息。 的人嗜中性白细胞吞噬体具有大约8.5 20-30分钟吞噬5后的碱性pH值。这意味着在NOX另外的非质子离子通道的存在2诱导的电荷补偿，作为融合和的酸性颗粒和鞋底补偿由HVCN1的内容物的释放将维持酸性环境，而相比之下，所观察到的。离子的移动，以补偿该负电荷也可经由渗透施加在吞噬体大小的变化。这些存在于高生理浓度的中性粒细胞可以是离子:钾离子已经显示出移动到吞噬体6，和氯化物离子的运动是另一个候选为嗜中性粒细胞功能7重要。 pH值在吞噬体的调节是用于抗微生物蛋白酶活性5是至关重要的。髓过氧化物酶（MPO）似乎具有最佳活性在pH 6，而对于组织蛋白酶G和弹性蛋白酶的最佳水平为pH 7-9和pH 8-10，分别为5。因此，在吞噬体的pH瞬时变化可以针对不同的酶，以提供有趣的活动龛ction。了解pH值如何参与嗜中性粒细胞杀死微生物可以提供新颖的嗜中性粒细胞增强微生物剂的设计的有用信息。 中性粒细胞吞噬体是一种高活性的环境。这使得难以准确评估pH值，因为染料可能容易被氧化，从而导致技术假象。从历史上看，异硫氰酸荧光素（FITC）已选择的染料来测量细胞内pH 8,9。然而，也有一些缺点为其测量中性粒细胞吞噬小体的pH使用。它具有6.4 10的pKa，这意味着它只能精确地被用于pH水平评估为5至7.5 8，因为它饱和在pH &lt;8 11。由于嗜中性粒细胞吞噬体pH值可以变得更加碱性5，FITC不能捕获全面的潜在的pH变化。进一步signifi嗜中性粒细胞中的上下文中倾斜问题用FITC是，它被认为是由MPO 12光漂白。的MPO抑制剂，叠氮化钠，可以用来限制光漂白13，但它已经表明，叠氮化钠直接降低了pH值吞噬体中的MPO无关的方式，并因此在这样的测定5不当使用。 相对于其他细胞内的染料，S-1具有7.5 10相对高的pKa。在酸性条件下，该分子是质子化的并且当在488纳米或以上激发产生560和600纳米之间的发射信号。当在分子中更多的碱性条件下脱质子化，发射波长为600纳米。在这两个波长的荧光强度的比例表示发光移，这是更比单一荧光测量可靠，因为它是由荧光团浓度和细胞结构的影响。 S-1可以缀合至抗原性材料，例如酵母聚糖14，虽然热灭活（HK） 白色念珠菌是优选的，因为较大的表面积给出了更一致的荧光读数。 我们也使用这种方法的修改来研究pH值随时间的变化（ 图3）5。此胞质pH值的测定方法可容易地应用于其它类型的细胞中，与多种碱性吞噬体14中别处描述15，16，和细胞。

<table><tbody><tr><td>&lt;em&gt;Candida albicans&lt;/em&gt; ATCC 10231 Vitroids&amp;nbsp; 80 CFU</td><td>Sigma-Aldrich</td><td>RQC14003-10EA</td><td>Place directly on a spread agar plate according to the manufacturer&#039;s instructions</td></tr><tr><td>YPD broth</td><td>Sigma-Aldrich</td><td>Y1375</td><td>Follow manufacturer&#039;s instructions, various providers exist</td></tr><tr><td>Dextran Clinical grade MW = 200,000-300,000</td><td>MP Biomedicals</td><td>2101514</td><td>Use gloves&amp;nbsp;</td></tr><tr><td>Heparin Sodium solution for infusion (preservative free), 1,000 IU/mL&amp;nbsp;</td><td>Fannin, Wockhardt UK Ltd</td><td>FP1077</td><td>We purchased from UCH inpatient pharmacy</td></tr><tr><td>SNARF-1 Carboxylic Acid, Acetate, Succinimidyl Ester - Special Packaging</td><td>Thermo Scientific/Invitrogen</td><td>SS22801</td><td>Use gloves</td></tr><tr><td>5-(and-6)-Carboxy SNARF-1, Acetoxymethyl Ester, Acetate</td><td>Thermo Scientific/Invitrogen</td><td>C1272</td><td>Use gloves</td></tr><tr><td>Vivaglobin human IgG&amp;nbsp; 160 mg/mL solution</td><td>CSL Behring</td><td>received as a gift</td><td>Various providers exist</td></tr><tr><td>Normal mouse serum</td><td>Abcam</td><td>ab7486</td><td>Various providers exist</td></tr><tr><td>Lymphoprep (density gradient medium)</td><td>Alere Ltd</td><td>1114547</td><td>Keep sterile and away from direct sunlight, can be substituted by Ficoll-Paque</td></tr><tr><td>Nigericin</td><td>Sigma-Aldrich</td><td>N7143</td><td>Toxic, use gloves, various providers exist</td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>47036</td><td>Toxic, use gloves, various providers exist</td></tr><tr><td>Dimethyl sulphoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418</td><td>Toxic, use gloves, 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>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>Magnesium sulphate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>M7506</td><td>Various providers exist</td></tr><tr><td>Potassium dihydrogen phosphate (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>P9791</td><td>Various providers exist</td></tr><tr><td>Calcium chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>C1016</td><td>Various providers exist</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>D9434</td><td>Various providers exist</td></tr><tr><td>Absolute Ethanol&amp;nbsp;</td><td>Sigma-Aldrich</td><td>2860</td><td>Various providers exist</td></tr><tr><td>Diphenylene iodonium (DPI)</td><td>Sigma-Aldrich</td><td>D2926</td><td>Various providers exist</td></tr><tr><td>Zinc chloride</td><td>Sigma-Aldrich</td><td>208086</td><td>Various providers exist, do not dilute in phosphate based buffers as it will precipitate</td></tr><tr><td>Poly L lysine</td><td>Sigma</td><td>P4707</td><td>Various providers exist</td></tr><tr><td>Laser scanning confocal microscope (Zeiss LSM 700)</td><td>Carl Zeiss</td><td></td><td>The microscope must be able to excite at 555 nm and measure emission at two channels simultaneously (560-600 nm, &gt;610 nm)</td></tr><tr><td>Ibidi &amp;micro; slide 8 well ibiTreat</td><td>Thistle Scientific</td><td>IB-80826</td><td>Other appropriate imaging dishes can be substituted</td></tr><tr><td>Soniprep 150</td><td>MSE</td><td></td><td>Any appropriate sonicator will suffice</td></tr><tr><td>TC20 Automated Cell Counter</td><td>BioRad</td><td></td><td>Various providers exist</td></tr><tr><td>1.5 mL Protein LoBind Tubes</td><td>Sigma-Aldrich</td><td>Z666505-100EA</td><td>Specially used as &lt;em&gt;Candida&lt;/em&gt; can stick to normal eppendorfs</td></tr></tbody></table>

imaging the neutrophil phagosome and cytoplasm using a ratiometric ph indicator

中性粒细胞是宿主先天防御的关键，因此，构成医学研究的一个重要领域。吞噬，细胞内的车厢中吞噬颗粒的杀戮和消化发生，对于那些需要精确调节中性粒细胞杀灭病原体的主要舞台。吞噬体的pH是仔细控制，反过来调节抗微生物的蛋白酶活性的一个方面。许多荧光pH敏感的染料已被用于可视化的吞噬体环境。 S-1具有比其它pH敏感染料，包括它的双发射光谱，其光漂白性和其高的pKa几个优点。使用这种方法，我们已经表明，中性粒细胞吞噬是相较于其他吞噬细胞异常碱性。通过使用染料的不同生物化学缀合，吞噬体可以从细胞质划定使得在吞噬体的大小和形状的变化进行评估。这使得˚F或进一步的监测离子的运动。

图1表示不同来源的嗜中性粒细胞的快照以证明不同吞噬体环境。为了缓解定量分析，种子孔与适当的细胞数是很重要的:太多会导致细胞层在彼此之上，这使得难以精确地查看封闭吞噬体;太少的意志，当然，提供较少的结果，尤其是因为并非每个中性粒细胞吞噬会。 图2是是过饱和的图像;红色点表示已经检测最大荧光 - 这可以通过分割图像的两个信道之间（使用材料清单或等效的建议的显微镜软件）进行评估。这可以通过减小激光的强度来抵消。使用各种缓冲系统的校准曲线示于图3中 ，改编自Levine 等。 5 &lt;/ SUP&gt;。误差线表明，在读数之间的荧光一些变化。 图4给出了如何针对吞噬体的pH值和区域中的数据可以被呈现的示例。这种方法允许以一个过铺设箱线图显示每个单独的测量。但是，这些数据也可以被显示的直方图条形图英寸<img alt="图1" src="/files/ftp_upload/55107/55107fig1.jpg" /> 图1:来自人和小鼠的嗜中性粒细胞的适当的快照图像。最右边是对应于pH值S-1染色的吞噬体的近似颜色的定性视觉系。黄色表示还有更多的酸度，而红色表示多种碱。 （A）示出了Hvcn1 - / -小鼠骨髓嗜中性粒细胞的吞噬作用后20分钟。吞噬体显得很红，碱性和肿胀。在的右下部分的红色箭头图像点到细胞内念珠菌 ，而在左上点到细胞外的粒子的箭头。 （B）示出了已摄取念珠菌的野生型小鼠骨髓嗜中性粒细胞;他们比Hvcn1碱性少得多- / -细胞。 （C）示出了在后的吞噬作用的相同点的人外周血嗜中性粒细胞。他们似乎比野生型小鼠细胞稍微碱性，但吞噬体仍然没有大又红的Hcvn1 - / -细胞。 （D）示出了在5μM的二亚苯基碘（DPI）存在下已吞噬念珠菌人中性粒细胞。所有的吞噬体是非常酸性的，pH为6或更小;药物抑制NADPH氧化酶，所以没有补偿性离子运动。从该熔合到吞噬体酸性颗粒释放质子导致酸性pH 20，并增加招募在V-ATP酶的至TH在用DPI 9处理E吞噬体膜。细胞质还与来自其他条件的细胞显得更为碱性。 <a href="http://ecsource.jove.com/files/ftp_upload/55107/55107fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55107/55107fig2.jpg" /> 图2:过饱和的Hvcn1 - / -小鼠骨髓中性粒细胞。它从其中的荧光数据是过饱和的解析图像排除是很重要的。如在第5.3节描述的那样，合成图像被分成两个图像（左上，红色箭头）与单独呈现两个通道。在该软件，范围指示器上选中（左下角，红色箭头），则它们是过饱和的任何像素是明亮的红色。有本过饱和在两个信道（1和2）。一个MAGNIF一些细胞的ication和胞外念珠菌显示在右下方，用箭头指向受影响的区域。分析这些点会导致错误的比例测量。 <a href="http://ecsource.jove.com/files/ftp_upload/55107/55107fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55107/55107fig3.jpg" /> 图3:S-1的荧光比率至pH测量转换标准校准曲线。 念珠菌独自在不同的缓冲液的标准曲线（标记为"TRIS"），并且当细胞用皂苷（"皂苷"）透化是非常相似的，显示出在S-1的内部和读出该单元（吞噬小体和胞外外介质）是相当的。误差条代表平均值±SD。当测试i中的S-1的比例/ pH值曲线被缩短n中的细胞质（"细胞质"），其应该被考虑在图像分析。该图已被从Levine 等改性。 5。 <a href="http://ecsource.jove.com/files/ftp_upload/55107/55107fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55107/55107fig4.jpg" /> 图4:吞噬体的pH值和区域的定量。该图表示数据呈现的一个例子。示出了用于吞噬体比率和对应的pH值（ - / - Hvcn1小鼠骨髓嗜中性粒细胞），B::使用编程软件R. A中生成的图的野生型小鼠骨髓嗜中性粒细胞，C:人末梢血嗜中性粒细胞，和D:人嗜中性粒细胞用5μMDPI N =300分之3。单个测量被示出为小方块，与中介的重叠箱线图一和四分位数间距。红色条代表的意思。如在图1中的图像中看到，Hvcn1 - / -细胞具有非常碱性吞噬体相比野生型小鼠和人嗜中性粒细胞。它们还具有更大的吞噬体区域（ 图4B中，n =300分之3）。人中性粒细胞吞噬体稍微比野生型小鼠嗜中性粒细胞更碱性大，同时用DPI孵育的人中性粒具有非常酸性和小吞噬体。 <a href="http://ecsource.jove.com/files/ftp_upload/55107/55107fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table><tbody><tr><td colspan="4"> 平衡盐溶液（BSS）缓冲 </td></tr><tr><td>氯化钠</td><td> 156毫</td><td></td><td></td></tr><tr><td>氯化钾</td><td> 3毫米</td><td></td><td></td></tr><tr><td> 硫酸镁 </td><td> 2mM的</td><td></td><td></td></tr><tr><td> KH 2 PO 4 </td><td> 1.25毫米</td><td></td><td></td></tr><tr><td> 氯化钙 </td><td> 2mM的</td><td></td><td></td></tr><tr><td>葡萄糖</td><td> 10毫</td><td></td><td></td></tr><tr><td> HEPES </td><td> 10毫</td><td></td><td></td></tr><tr><td colspan="4">用NaOH或HCl pH 7.4的</td></tr><tr><td colspan="4"> YPD肉汤 </td></tr><tr><td> YPD肉汤</td><td> 50克</td><td></td><td></td></tr><tr><td>蒸馏水</td><td> 1升</td><td></td><td></td></tr><tr><td colspan="4">高压釜在121℃下15分钟</td></tr><tr><td colspan="4">对于YPD琼脂:高压灭菌之前添加15g / L的琼脂</td></tr><tr><td colspan="4"> 10％葡聚糖溶液 </td></tr><tr><td>葡聚糖临床级</td><td></td><td> 50克</td><td></td></tr><tr><td>氯化钠</td> &lt;TD&gt; </td><td> 4.5克</td><td></td></tr><tr><td>蒸馏水</td><td></td><td> 500毫升</td><td></td></tr><tr><td colspan="4">添加到玻璃瓶中，高压釜在121℃下15分钟</td></tr><tr><td colspan="4"> 2X盐溶液 </td></tr><tr><td>氯化钠</td><td></td><td> 18克</td><td></td></tr><tr><td>蒸馏水</td><td></td><td> 1升</td><td></td></tr><tr><td colspan="4">添加到玻璃瓶中，高压釜在121℃下15分钟</td></tr><tr><td colspan="4"> 10％皂素股票 </td></tr><tr><td> BSS缓冲</td><td></td><td> 50毫升</td><td></td></tr><tr><td>皂素</td><td></td><td> 5克</td><td></td></tr><tr><td colspan="4">热BSS缓冲至37℃，加入皂苷和混合。 </td></tr><tr><td colspan="4">在4℃加入0.1％叠氮化钠作为防腐剂，存储混合物。 </td> &lt;/ TR&gt; <tr><td colspan="4"> 校正缓冲液 </td></tr><tr><td colspan="4"> 念珠菌标准曲线 </td></tr><tr><td colspan="4">首先弥补0.15M的股票每个缓冲区的</td></tr><tr><td colspan="4">弥补了0.15M的NaCl溶液</td></tr><tr><td colspan="4"> 15mL的最终体积:5mL的期望的缓冲溶液的0.15 M + 10毫升0.15M的NaCl溶液</td></tr><tr><td> pH值3 </td><td> 100mM NaCl的</td><td> 50mM的甘氨酸</td><td></td></tr><tr><td> pH值4 </td><td> 100mM NaCl的</td><td> 50mM乙</td><td></td></tr><tr><td> pH值5 </td><td> 100mM NaCl的</td><td> 50mM乙</td><td></td></tr><tr><td> pH为6 </td><td> 100mM NaCl的</td><td> 50mM乙</td><td></td></tr><tr><td> pH值7 </td><td> 100mM NaCl的</td><td>的50mM Tris </td><td></td></tr><tr><td> pH值8 </td><td> 100mM NaCl的</td><td>的50mM Tris </td><td></td></tr><tr><td> pH值9 </td><td> 100mM NaCl的</td><td>的50mM Tris </td><td></td></tr><tr><td> pH值10 </td><td> 100mM NaCl的</td><td> 50mM的甘氨酸</td><td></td></tr><tr><td> pH值11 </td><td> 100mM NaCl的</td><td> 50mM磷酸盐</td><td></td></tr><tr><td> pH值12 </td><td> 100mM NaCl的</td><td> 50mM磷酸盐</td><td></td></tr><tr><td> pH值13 </td><td> 100mM NaCl的</td><td> 50mM磷酸盐</td><td></td></tr><tr><td colspan="4"> 胞浆标准曲线 </td></tr><tr><td colspan="4">使用先前作出的0.15M股票的缓冲溶液</td></tr><tr><td colspan="4">弥补了0.15M的KCl溶液</td></tr><tr><td colspan="4"> 15mL的最终体积:5mL的期望的缓冲液+ 10毫升0.15M的KCl溶液的0.15M的</td></tr><tr><td colspan="4">在乙醇尼日利亚菌素的10mM储备，添加15μL到每个终体积溶液</td></tr><tr><td> pH值3 </td><td> 100mM的氯化钾</TD&gt; <td> 50mM的甘氨酸</td><td> 10μM尼日利亚</td></tr><tr><td> pH值4 </td><td> 100mM的氯化钾</td><td> 50mM乙</td><td> 10μM尼日利亚</td></tr><tr><td> pH值5 </td><td> 100mM的氯化钾</td><td> 50mM乙</td><td> 10μM尼日利亚</td></tr><tr><td> pH为6 </td><td> 100mM的氯化钾</td><td> 50mM乙</td><td> 10μM尼日利亚</td></tr><tr><td> pH值7 </td><td> 100mM的氯化钾</td><td>的50mM Tris </td><td> 10μM尼日利亚</td></tr><tr><td> pH值8 </td><td> 100mM的氯化钾</td><td>的50mM Tris </td><td> 10μM尼日利亚</td></tr><tr><td> pH值9 </td><td> 100mM的氯化钾</td><td>的50mM Tris </td><td> 10μM尼日利亚</td></tr><tr><td> pH值10 </td><td> 100mM的氯化钾</td><td> 50mM的甘氨酸</td><td> 10μM尼日利亚</td></tr><tr><td> pH值11 </td><td> 100mM的氯化钾</td><td> 50mM磷酸盐</td><td> 10μM尼日利亚</td></tr><tr><td> pH值12 </tD&gt; <td> 100mM的氯化钾</td><td> 50mM磷酸盐</td><td> 10μM尼日利亚</td></tr><tr><td> pH值13 </td><td> 100mM的氯化钾</td><td> 50mM磷酸盐</td><td> 10μM尼日利亚</td></tr><tr><td colspan="4"> pH值与任一盐酸或氢氧化钠所有校准溶液</td></tr></tbody></table> 表1:缓冲剂的组成。此表描述在协议中使用的不同的缓冲液的适当的组合物。 补充代码文件。该文件包含了许多宏，通过阿普·莱温写的，这是必要的图像分析。作者会很乐意尝试解决使用此代码相关的任何疑问。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/55107/Phagosomal_measurement.txt" target="_blank">请点击这里下载此文件。</a>

Watch this Scientific Journal Video about Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator at JoVE.com

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

这手稿描述了一种简单的方法来测量吞噬体的pH值和面积以及使用比率量度指示器seminaphthorhodafluor（SNARF）-1，或S-1人和小鼠嗜中性粒细胞的胞质pH值。这是使用活细胞共聚焦荧光显微镜和图像分析来实现的。

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 ...

imaging-neutrophil-phagosome-cytoplasm-using-ratiometric-ph

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

10.0K 次观看.  University College London. 这手稿描述了一种简单的方法来测量吞噬体的pH值和面积以及使用比率量度指示器seminaphthorhodafluor（SNARF）-1，或S-1人和小鼠嗜中性粒细胞的胞质pH值。这是使用活细胞共聚焦荧光显微镜和图像分析来实现的。 

视频: Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

The development of fluorescent indicators represented a revolution for life sciences. Genetically encoded and synthetic fluorophores with sensing abilities allowed the visualization of biologically relevant species with high spatial and temporal resolution. Synthetic dyes are of particular interest thanks to their high tunability and the wide range of measureable analytes. However, these molecules suffer several limitations related to small molecule behavior (poor solubility, difficulties in targeting, often no ratiometric imaging allowed). In this work we introduce the development of dendrimer-based sensors and present a procedure for pH measurement in vitro, in living cells and in vivo. We choose dendrimers as ideal platform for our sensors for their many desirable properties (monodispersity, tunable properties, multivalency) that made them a widely used scaffold for several biomedical devices. The conjugation of fluorescent pH indicators to the dendrimer scaffold led to an enhancement of their sensing performances. In particular dendrimers exhibit reduced cell leakage, improved intracellular targeting and allow ratiometric measurements. These novel sensors were successfully employed to measure pH in living HeLa cells and in vivo in mouse brain.

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

Fluorescence sensors are powerful tools in life science. Here we describe a methodology to synthesize and use dendrimer-based fluorescent sensors to measure pH in living cells and in vivo. The dendritic scaffold enhances the properties of conjugated fluorescent dyes leading to improved sensing properties.

合成，细胞和交货<em&gt;在体内</em&gt;的树状为基础的pH值传感器应用

The development of  fluorescent indicators represented a revolution for life sciences. Genetically  encoded and synthetic fluorophores with sensing ...

科研

化学

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 ...

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 ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

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 ...

Ratiometric Imaging of Extracellular pH in Dental Biofilms

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 &#181;m thickness, but is limited to the pH range between 4.5 and 7.0.

A pH-sensitive ratiometric dye is used in combination with confocal laser scanning microscopy and digital image analysis to monitor extracellular pH in dental biofilms in real-time.

在口腔生物膜细胞外pH值成比例成像

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites ...

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 ...

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

用荧光基因编码的 ph 指示剂对果蝇氏小管上皮细胞内 ph 值的光学定量

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...