We thank Dr. Gilles Dupuis for critical reading of the manuscript. 
Funding
This work was supported by the Canadian Institutes for Health Research, grant # MOP-126173. C.M.D is a member of the Fonds de la Recherche en Sant&#233; (FRSQ)-funded Centre de Recherche Clinique &#201;tienne LeBel.

1，准备解的蜂窝pH校准<ol><li>加入下列化合物五个单独的50ml锥形管中，制备5解决方案，以用于校准: <ul><li>氯化钠（1毫升1M的NaCl）（1 M氯化钠=0.58克在10ml H 2 O） </li><li>氯化钾（6.75毫升1M的KCl）（1摩尔KCl =0.75克在10ml H 2 O） </li><li>葡萄糖（1毫升1 M D-葡萄糖）（1M D-葡萄糖=1.80克在10毫升H 2 O） </li><li>用MgSO 4（0.05毫升1M 硫酸镁 ）（1M，用MgSO 4 =1.20克在10ml H 2 O） </li><li> 20毫米肝素钠（1毫升1M的HEPES）（1 M羟乙基=2.38克在10毫升H 2 O） </li><li> 氯化钙 （0.05毫升，浓度1M氯化钙2）（1米氯化钙2 =1.47克在10毫升H 2 O） 准备好所有的解决方案在双蒸水 （双蒸2 O）。 </li></ul></li><li>完成每个溶液用35ml双蒸2 O中并用磁力搅拌器混合，直到这样率被实现。 </li><li>调整各溶液的pH用1N KOH或1N HCl中，得到5.5，6.0，6.5，7.0和7.5的pH值。然后，调整与去离子水的体积为50毫升的最终体积。 </li><li>措施5毫克尼日利亚菌素，并将其添加到670μL无水乙醇中制成10毫米尼日利亚菌素原液。 注意:尼日利亚菌素是剧毒的，必须非常小心（手套，安全眼镜和面罩）进行处理。通过倒置混合该溶液直到尼日利亚菌素完全溶解。 </li><li>加0.05 10mM的尼日利亚菌素（终浓度10μM）ml到每个校准溶液。尼日利亚菌素是一种离子载体，增加细胞膜的通透性，并导致细胞内和细胞外pH值的平衡中的细胞外K +去极化浓度的存在。 </li><li>校准解决方案，可存储长达1个月，在4℃ </li></ol> 2，细胞的制备<ol><li>下一个生物拉利Ý罩，种子细胞于35毫米×10毫米培养皿中的2ml补充有10％胎牛血清和抗生素的生长培养基。使用需要确保在24小时后约50％汇合的细胞的量。 注意:在我们的实验中，我们使用HT-1080细胞系和板每培养皿1×10 5个细胞。 </li><li>确定所需要的实验和板用于校准5个额外的培养皿培养皿的数量。 </li><li>地方培养皿在37℃/ 5％CO 2培养箱至少24小时。 </li></ol> pH值传感探头3。准备<ol><li>在DDH 2 0准备荧光pH探头8 -羟基芘-1，3,6三磺酸三钠盐（HPTS /羟基芘磺酸）的1M贮存液。此解决方案必须存储在RT及避光。 </li><li>准备1毫米5的原液 - （和6） - 羧基seminaphthorhodafluor乙酰氧基甲基醋酸酯（C-SNARF-1 AM;叫therea折后SNARF-1）通过在1.76毫升的DMSO中溶解1毫克SNARF-1。通过反复吹打调匀。 SNARF-1溶液必须储存在-20℃下并避光。 </li></ol> 4。细胞标记<ol><li>十六个小时之前活细胞成像，加入2微升1M HPTS原液（终浓度为1mM），以含2ml完全培养基中的每个培养皿中。 </li><li>返回培养皿的孵化器。该HPTS探头是细胞透性，因此对标签的细胞器探头必须采取由胞饮作用。这个条件要求孵育细胞与HPTS在补充有血清的培养基中进行。 </li><li>温育后用HPTS，用无菌PBS洗细胞两次，并加入2ml无血清培养基中至少2小时。孵育SNARF-1需要在无血清培养基中进行的，因为血清可以防止探针的吸收。 </li><li>加入10μl1毫米SNARF-1原液获得自动对焦5微米INAL浓度，使细胞孵育20分钟，在37℃，在昏暗的灯光下。 C-SNARF-1浓度和孵育时间可根据细胞类型而有所不同。 </li><li>在孵育结束时，在无菌PBS洗涤细胞两次，并加入2ml无血清培养基中。 </li></ol> 5，加入pH值调节剂的<ol><li>评估方法是否可以检测在细胞pH值的变化，可以使用该调节细胞内pH值不同的试剂: <ul><li>巴弗洛霉素A1，V型ATP酶5的特异性抑制剂</li><li>的Na + / H +交换体的同种型1（NHE-1）6 </li><li> NH 4 Cl等 ，弱碱，增加细胞内pH值7 </li></ul><ol><li>制备储备溶液上面提到的试剂: <ul><li>巴弗洛霉素A1:加10微克巴弗洛霉素A1至160微升的DMSO中，得到100μm的原液</li><li> EIPA:添加EIPA 25毫克至1.67 ml甲醇，obtai50毫米的Na终浓度</li><li> NH 4 Cl等 :溶解0.53克氯化铵在10毫升双蒸2 O，以获得1mM的终浓度。 </li></ul> 注:所有股票的解决方案必须保持在4℃，惟必须保存在-20℃，避光巴弗洛霉素A1。 </li><li>在单独的培养皿中，加入下述试剂之一: <ul><li> 2微升100μM的巴弗洛霉素A1（终浓度为100nM） </li><li> 1微升的50mM EIPA（终浓度为25μM） </li><li> 40微升1M 氯化铵 （终浓度为20毫摩尔） </li></ul></li><li>于室温孵育30分钟，在37℃下</li></ol></li><li>进行图像采集。 </li></ol>在活细胞成像显微镜6。规格及收购设置<ol><li>使用进行活细胞成像的40X物镜安装在一个频谱倒置扫描共聚焦microscoPE配有电动XY载物台，其容纳玻璃底培养皿中的环境控制（温度，湿度，CO 2）的阶段培养箱的腔室。 </li><li>这里所用的共聚焦仪（奥林巴斯FV1000）配备有: <ul><li>二极管激光器405 nm处</li><li> A 40毫瓦氩激光（458纳米，488纳米，515纳米） </li><li>氦氖激光（543纳米） </li><li>萤石10倍的目标，NA0.4 </li><li>一个LUCPLFLN 40X客观，NA0.60 </li><li>一个PLAPONSC计划APO 60X油浸物镜，NA1.4 </li><li>二向色镜DM 405/458/488/543 </li><li>一个SDM 560和640 SDM的二向色滤光片</li><li>一个BA655-755屏障过滤器</li><li>分束器BS20/80 </li><li>数据分析软件进行图像采集和分析， </li></ul></li><li>一旦孵化阶段已升温至37℃，并在显微镜设置，将以前与HPTS和SNARF-1孵育成共焦成像舞台腔培养皿。 </li><li>一折后定位感兴趣的区域，调整软件采集设置图1。 <ul><li>设置了两个虚拟通道组。 </li><li>启动对应于激发HPTS在505 405 nm，发射集合中的第一阶段 - 525 nm和SNARF-1在激发在570 543 nm，发射集合 - 600纳米。 </li><li>启动对应于激发HPTS在505 458 nm，发射收集第二阶段 - 525 nm和SNARF-1在激发在640 543 nm，发射集合 - 650纳米。 </li><li>共焦孔径设定为175微米的默认和最佳光圈需要手动设定为300微米。 </li><li>取图像通过堆叠串行水平光学部分0.4微米厚（800×800像素）。 </li></ul></li></ol>注:一个40X的目标是有用的同时观察几种细胞和行之有效的细胞器观看。然而，空间分辨率和信号强度是贝特使用60X石油目标Ř实现。此外，有两种染料之间没有激发或发射的串扰。 pH值传感探头7。校准<ol><li>校准之前，从预先用HPTS和SNARF-1孵育的培养皿中除去培养基，用无菌PBS洗涤两次，然后加入2毫升5校准溶液之一。 </li><li>将培养皿放入共焦载置台室。 </li><li>按照下面的设置进行时间推移图像采集: <ol><li>设置图像和间隔持续时间为实验的数目。 注意:我们通常使用的图像之间的1分钟时间长达30分钟。 </li><li>程序快门关闭采集每幅图像之间。 </li></ol></li><li>记录每分钟的荧光强度在一个电子表格软件，并确定时间时，荧光强度稳定。细胞内pH值是15后通常稳定 - 20分钟。 </li><li> Ř使用新的培养皿中其余的每个校准的解决方案EPEAT步骤7.1至7.4。 </li></ol> 8，数据采集<ol><li>一旦校准已经完成，从4-5含每场15-20细胞随机选择的领域取得的样本数据。两个图像的每个字段获得:1为HPTS标记（小泡）和第二对SNARF-1标记（胞质溶胶）。 </li><li>背景荧光（细胞外区域）的数字减去两个探针的荧光图像。标记的囊泡和细胞溶质从各自的存储图像是使用二进制掩码中选择。对于每个pH值传感探头，荧光强度的数值刻度（0-4,095灰度级规模）来测量。平均强度的细胞器或细胞浆的所有像素计算和值来计算荧光比率。 </li></ol> 9。数据分析<ol><li>各个软件包是市售的用于数据分析。 即Fluoview软件。 </li><li>在电子表格中，记录值，并计算荧光比率如下:在HPTS的情况下，R =（F 458纳米 /女性405 nm）和，在SNARF-1，R =（F 644纳米 /女性584纳米的情况下）。 </li><li>用科学绘图软件如的GraphPad棱镜绘制数据。情节应该描绘荧光比值（Y轴），pH值（X轴）的函数。曲线拟合使用非线性回归来实现。 </li><li>采集样品的荧光强度如步骤8.2中所述，并计算荧光比值。使用校准曲线转换成比例的pH值。 </li></ol>

<ol>
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No conflict of interest declared.

在不同的细胞区室的活细胞成像定量测量pH值是需要精确测量细胞对外部挑战pH值的变化。然而，这仍然是主要的障碍之一就是能够容易地和特异性靶向细胞区室。在这方面，很少有研究强调了使用的HPTS通过电穿孔或显微注射8-10导入细胞胞内pH指示剂。这些技术存在严重的缺点，即电穿孔引起广泛破坏细胞，显微注射法需要特殊的设备和技术技能。息的，Hinton 等人 ，以前曾报道说HPTS可以被细胞吸收通过胞饮作用的一个过程，SNARF-1和HPTS的组合可以被用来监视在细胞质和核内体/溶酶体区室3的pH值。我们已经使用这个属性来记录在溶酶体/内体compartm pH值的变化耳鼻喉科现场HT-1080细胞。证据表明，HPTS是位于该隔室，因此，可作为一种可靠的pH指示剂是由共染色用荧光团标记的转铁蛋白得到，溶酶体/核内体室的一个标记，或与嗜酸性染料大多标签溶酶体图2，另外，使用靶向不同的细胞区室的不同的细胞内pH值调节剂的表示，巴弗洛霉素A1特别增加囊内pH值，而不改变胞质pH值。与此相反，EIPA主要改变细胞内pH值，但对内涵体和溶酶体pH值很小或没有影响，而氯化铵的pH影响两个隔室中。这些结果提供了证据，SNARF-1和HPTS可以用于同时监测在这两种不同的细胞区室的pH值的变化。 这里描述的方法的一个重要特征是利用共焦激光扫描显微镜。大多数研究回购RTED在细胞内pH测量文学已经完成了荧光分光光度法。虽然易于使用，并提供快速的数据采集，这种方法不允许观察细胞的实验中。这个缺点可能成为重要意义，因为它一直是我们的经验，在校准步骤，在酸性条件下延长潜伏期（pH值5.5）或碱性（pH值8.0）条件下可能对不同细胞类型的影响，并触发细胞凋亡。在HT-1080细胞的情况下，细胞凋亡导致的红色自发荧光，从而与SNARF-1（数据未示出）的pH依赖性荧光干扰。为了解决这个问题，我们使用了不同的培养皿中每个校准和丢弃的实验中观察到细胞凋亡或其他细胞改变的迹象。利用共聚焦显微镜用于pH测量也使得数据分析更容易。例如，共聚焦荧光显微镜图像分析可以用于丢弃的工件，apoptotiC单元和背景噪音，使计算更容易，更准确。 尽管相对于其它方法这里描述的方法提供了许多优点，但是它有一些局限性。其中之一是涉及到需要为每个探针的不同加载条件。对于HPTS，染料，必须采取由胞饮作用。这种限制要求细胞必须维持一段延长的时期（ 例如 12小时），在无血清的培养基中，以促进胞饮作用，从而内吞室的标记。与此相反，细胞穿透物SNARF-1需要在无血清培养基中进行培养，以防止乙酰氧基甲酯（AM）的水解由含血清的非特异性酯酶的可能性。这些限制将排除那些需要加载不兼容的细胞培养条件的实验方法。此外，HPTS的酸性介质中的灵敏度是一个重要的限制。 如图2，F兴奋在458 nM的HPTS的luorescence强度显示的变化最小，在pH 6.0或更低，与接近背景值的比率。 HPTS的这种内在的属性应该被考虑的非常酸性的细胞区室，如具有的pH值低至4.0至5.0溶酶体研究的情况。然而，就我们所知，HPTS是唯一可用的染料比例可以被用来测量内体内的pH值。例如，常用的lysosensor染料将更加酸性溶酶体内大多分区。这种染料的分区的内涵体隔室中的程度将取决于pH值下这些囊泡内，这是一个主要的缺点测定膀胱内pH值在细胞中，其中内体pH平衡被改变时。 总之，我们在这里描述的方法在单细胞同时测量胞质和核内体的pH值。这是用于pH稳态的细胞中的研究尤为重要WHERË突变或药物可影响胞内体流出和质子的涌入。

<table border="0" cellpadding="0" cellspacing="0" width="872">
	<tbody>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">Solutions for calibration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Sodium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">L-11621</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Potassium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M-12445</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">D-Glucose (dextrose)</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">D16-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Magnesium sulfate</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M65-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">HEPES</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Calcium chloride dihydrate</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M-1612</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Deionized water</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Nigericin</td>
			<td style="width:136px;">Calbiochem</td>
			<td align="right" style="width:119px;">481990</td>
			<td>Toxic by inhalation or in contact with skin</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td style="width:315px;">Keep protected from the light at 4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Culture dishes &#8709; 35mm</td>
			<td style="width:136px;">BD Biosciences</td>
			<td align="right" style="width:119px;">354456</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">pH-sensing probes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS)</td>
			<td style="width:136px;">Life technologies</td>
			<td style="width:119px;">H-348</td>
			<td>Keep away from the light at room temperature</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">5-(and-6)-Carboxy SNARF&#174;-1, Acetoxymethyl Ester, Acetate</td>
			<td style="width:136px;">Life technologies</td>
			<td style="width:119px;">C-1271</td>
			<td>Keep away from the light and humidity at -20&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Dimethyl Sufoxide (DMSO)</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">BP231-1&#160;</td>
			<td>Harmful</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">Phosphate buffered saline (PBS) 100X</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Potassium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M-12445</td>
			<td>20 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Sodium phosphate monobasic</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">S369-1</td>
			<td>115 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Potassium phosphate monobasic</td>
			<td style="width:136px;">J.T Baker</td>
			<td style="width:119px;">3246-01</td>
			<td>20 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Deionized water</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>Bring to 1 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Autoclave and adjust pH to 7.4</td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">Phosphate buffered saline (PBS) 1X</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">PBS 100X</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>50 mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Sodium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">L-11621</td>
			<td>40,5 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Deoinized water</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>Bring to 5 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">pH modulators</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">&#160;Bafilomycin A1</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:119px;">B-1793</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-(N-Ethyl-N-isopropyl)amiloride (EIPA)</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:119px;">A3085</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Ammonium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">A649-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

对于pH值在细胞内和细胞内体/溶酶体细胞区室的同时测量，SNARF-1的比率荧光pH值传感探头和HPTS被使用。 SNARF-1被限制在细胞内隔室而HPTS允许内体/溶酶体区室的成比例的pH测量。小区负荷与HPTS 16小时允许在内涵体/溶酶体4的选择性定位。荧光从装入的pH传感探头HT-1080细胞中记录的一个典型例子示于图2A中 。证据表明，HPTS标签既内涵体和溶酶体区室是由共染色得到的与Alexa 546缀合的转铁蛋白（胞内体）或荧光lysotrophic探针（Lysotracker深Reddye）。数据清楚地表明，HPTS染色的两个隔室图2A中 。 使用上述的方法，在活细胞中的细胞内pH值下使用，测定每个探针图2B和2C的校准曲线的基础上，各pH值敏感的染料的比例特性。五个校准溶液用于荧光强度转换为pH单位。从三个独立的校准数据报告每个pH值传感探头图2C。每个数据点代表荧光强度在四百〇五分之四百五十八纳米的HPTS或在给定的pH值五百八十四分之六百四十四纳米的SNARF-1的发射波长的激发波长的比值。校准曲线为两个探针显示出这是最好装有一个指数方程图2C非线性关系。该方法用于检测细胞区室相关的pH值的变化的效率，用NH 4 Cl等 ，弱碱，增加细胞内pH值，巴弗洛霉素A1，V型ATP酶和EIPA药理抑制剂，NHE-活HT-1080细胞的评价1抑制剂。 NH 4 Cl等触发囊内和胞质solic pH值增加从6.35至7.10和7.00至7.40，分别如图3A和3B。巴弗洛霉素A1诱导内体溶酶体车厢的碱化不改变细胞内pH值。与此相反，EIPA诱导的细胞溶质达到6.62的pH值相比，在未经处理的细胞图3A和3B 7.00的酸化。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51395/51395fig1highres.jpg" width="500" /> 图1为共聚焦激光扫描显微镜获得的设置图。 （一）第一阶段被设置为激发HPTS在405 nm和SNARF-1在543 nm处同时通过分色（DM405/488/543/633）镜子。荧光发射是利用分色镜（SDM560）首次录得HPTS在505-525纳米。 SNARF-1的荧光发射，然后通过一个bandpa监视SS干涉滤光片在570-600 nm范围内。（二）第二阶段设置为同时收集HPTS的荧光发射（EXC，458 nm）和SNARF-1（极好，543纳米）。由激光束产生的激励探头通过一个分束器（BS20/80）和荧光发射每个探头是通过在第一阶段使用的分色镜收集。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51395/51395fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51395/51395fig2highres.jpg" width="500" /> 图2 在 pH值传感探头的现场校准。 （一）HPTS和SNARF-1亚细胞定位在HT-1080纤维肉瘤细胞的代表性图像。上的图像显示，SNARF-1标记细胞质，而HPTS-相关的荧光显示囊内定位。 HT-1080细胞培养30分钟用Alexa 546标记的转铁蛋白（5微克/毫升）或lysotrophic染料（Lysotracker深Reddye）（100纳米），分别为。下部的图像显示，HPTS共定位与转铁蛋白和lysotrophic染料（60X）（B）两者的pH传感探头的细胞温育15分钟以校准溶液在pH 6.0和pH 7.5代表性图像（C）荧光强度的关系使用校准溶液pH值的函数确定HPTS和SNARF-1的比率。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51395/51395fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" fo:content-width="3in" src="/files/ftp_upload/51395/51395fig3highres.jpg" width="300" /> 图3。调制nt的我racellular pH值通过巴弗洛霉素A1，EIPA与NH 4 Cl。药理抑制剂（巴弗洛霉素A1，EIPA）和细胞内pH值调节剂的NH 4 Cl等被用来验证的方法。图表显示记录在内涵体/溶酶体区室（A）或装有HPTS和SNARF-1，并孵育在氯化铵的存在或不存在（对照）30分钟HT-1080细胞的细胞质中（B）（pH值下20毫米），进出口许可证法（25微米）或巴弗洛霉素A1（100纳米）。数据表示为平均值±SEM（** P 0.005，*** P 0.0001）。

Watch this Scientific Journal Video about Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy at JoVE.com

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

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 ...

simultaneous-ph-measurement-endocytic-cytosolic-compartments-living

Nanyang Technological University

Research

JoVE Journal

Biology

14.9K 次观看.  Université de Sherbrooke. 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. 

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

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

影像生活细胞的形状变化果蝇胚胎

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

科研

生物学

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

基于荧光测定活细胞中的钙进入商店经营:从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH are highly regulated in yeast cells and occupy a central role in overall pH homeostasis. We describe protocols for ratiometric measurement of pH in vivo using pH-sensitive fluorophores localized to the vacuole or cytosol. Vacuolar pH is measured using BCECF, which localizes to the vacuole in yeast when introduced into cells in its acetoxymethyl ester form. Cytosolic pH is measured with a pH-sensitive GFP expressed under control of a yeast promoter, yeast pHluorin. Methods for measurement of fluorescence ratios in yeast cell suspensions in a fluorimeter are described. Through these protocols, single time point measurements of pH under different conditions or in different yeast mutants have been compared and changes in pH over time have been monitored. These methods have also been adapted to a fluorescence plate reader format for high-throughput experiments. Advantages of ratiometric pH measurements over other approaches currently in use, potential experimental problems and solutions, and prospects for future use of these techniques are also described.

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).

液泡和胞质pH值的测量<em&gt;在体内</em在酵母细胞悬浮液

Vacuolar and  cytosolic pH are highly regulated in yeast cells and occupy a central role in  overall pH homeostasis. We describe  protocols for ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

使用4D共聚焦显微镜分析颅面形态的斑马鱼

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

在肾小管上皮细胞胞浆钙的测量流式细胞仪

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

线粒体钙和线粒体膜的同时测量通过荧光显微镜活细胞的潜在

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.