我们感谢希尔丝廷Elgass博士和莫纳什显微成像技术援助萨拉信条博士和威康信托基金会和医学研究理事会英国的财政支持。 MMcK支持澳大利亚研究理事会未来的奖学金计划（FT120100459），威廉·巴克兰基金会，澳大利亚线粒体病基金会（AMDF），医学研究和莫纳什大学的哈德逊研究所。这项工作是由维多利亚州政府经营性基础设施支持计划的支持。

1.细胞的制备<ol><li>生长10 1cm池培养皿的细胞或75cm 2的烧瓶中补充有5％（体积/体积）胎牛血清（FBS）和1×青霉素/链霉素（P / S培养基[10毫升Dulbecco氏改良的Eagles培养基（DMEM）中）]，在37℃/ 5％CO 2。 </li><li>收获细胞，通过抽吸除去培养基，然后用5ml 1×磷酸盐缓冲盐水（1×PBS）洗涤。通过抽吸除去1×PBS中，然后加入1.5毫升0.25％（重量/体积）胰蛋白酶/ 0.25％（重量/体积）乙二胺四乙酸（EDTA），并在37℃/ 5％CO 2孵育2分钟。点击菜或烧瓶轻轻除去细胞，然后悬浮在5毫升培养基。 </li><li>加入12微升悬浮细胞到血细胞计数器的细胞。 </li><li>板块细胞菜肴或共焦成像腔盖玻片。 注意:这些将有一个合适的塑料或玻璃底已设计用于倒置显微镜的使用。 </li><li>板细胞，使日在〜80％汇合上成像的天来实现。例如，大约2×10 4个 143B骨肉瘤细胞可以接种到成像之前的8孔腔室载玻片一天一个阱。 </li><li>孵育细胞过夜，在37℃/ 5％CO 2，以允许细胞附着和恢复。 </li></ol> 2.缓冲器，用于TMRM和荧光 - 4数字影像<ol><li>制备含有156 mM氯化钠，3毫米氯化钾，2mM的硫酸镁 ，记录溶液（RS） - 1.25毫KH 2 PO 4，10mM的D-葡萄糖，2mM的氯化钙和10mM的HEPES pH值7.35。 RS储存在-20ºC等分。 </li><li>制备细胞内中等（IM）含有6毫摩尔NaCl，130毫摩尔的KCl，7.8毫的MgCl 2，1mM的KH 2 PO 4，0.4mM的氯化钙 ，2毫EGTA，10mM的HEDTA，2毫苹果酸盐，2mM的谷氨酸，2毫ADP和20mM HEPES pH值7.1。苹果酸盐，谷氨酸盐和ADP的的200mM储备溶液可以单独制备，等分并储存在-20℃。这些STocks可以只使用前加入到IM。 </li><li>从商业供应商的 Ca 2+游离Hank氏缓冲盐溶液（HBSS）中，可以得到的预制备。添加乙二醇 - 双（β氨基乙基醚）-N，N，N&#39;，N&#39;-四乙酸（EGTA），以500μM的最终浓度。 </li><li>制备在100％甲醇的10mM四甲甲酯，高氯酸根（TMRM）的贮备溶液。从这种股票使2μm的工作原液在蒸馏水H 2 O </li><li>制备的10mM维拉帕米在100％乙醇中的储备溶液。 </li><li>制备为1mg / ml（重量/体积）的二甲亚砜（DMSO）的Fluo-4乙酰氧基甲基酯（的Fluo-4，AM）的储备溶液。 注:荧光- 4，AM是在结合钙离子表现出增加的荧光钙指标。的Fluo-4是FLUO-3的类似物，由氟取代的两个氯取代基。这导致在488纳米增加的荧光激发和更高的荧光信号水平。该AM酯基团产生的是能够渗透细胞膜不带电的荧光 - 4分子。一旦进入细胞内，在AM酯通过非特异性酯酶裂解，导致在被截留在细胞内的Fluo-4的充电形式。 </li><li>制备在100％乙醇1mM的羰基氰化物对 - trifluoromethoxyphenylhydrazone（FCCP）的贮备溶液。 </li><li>制备的25毫克/毫升的蒸馏水H 2 O储备溶液（重量/体积）毛地黄皂苷</li><li>制备100μM的毒胡萝卜素的DMSO储备溶液。 </li><li>在蒸馏H 2 O制备的40毫氯化钙（氯化钙2）的储备溶液</li></ol> 3. TMRM和荧光 - 4，AM细胞染色<ol><li>用20nM的TMRM制备RS染色溶液，5微克/毫升（重量/体积）的Fluo-4，AM和0.005％表面活性剂，如尼克F-127的RS。 注:这种表面活性剂是一种非离子型多元醇，其促进的Fluo-4，AM的溶解。注:也可加入10μM的维拉帕米到INHIBI吨TMRM出口通过质膜药转运如果它是在细胞类型中表达所分析。 </li><li>通过吸移管除去从细胞培养基，并用100微升1×PBS洗涤。 </li><li>通过吸移管除去1×PBS中并孵育在250微升的RS染色溶液的细胞在室温下45分钟。 注意:一旦的Fluo-4，AM进入细胞中，AM酯通过蜂窝酯酶裂解。在室温下温育抑制酯酶的活性，允许的Fluo-4，AM进入线粒体。 </li><li>通过吸移管除去RS染色液和洗涤细胞在100μl 的 Ca 2+的HBSS以除去过量的Fluo-4，AM和TMRM。 </li><li>用25微克/毫升制备的IM成像溶液（重量/体积）毛地黄皂苷，200nM的TMRM和在IM1μM的毒胡萝卜素。 注:毛地黄皂苷的浓度可能需要进行调整，以确保线粒体内膜不会发生的那透。这可凭经验通过评估强度来确定在不同浓度的毛地黄皂苷的TMRM信号。 </li><li>用移液管除去的 Ca 2+的HBSS，并添加300微升的IM成像溶液到细胞中。离开细胞在室温下平衡10分钟。细胞现在已经准备好与氯化钙补充成像。 </li></ol> 4.细胞成像<ol><li>将菜或在IM成像解决方案细胞腔盖玻片上倒置激光扫描共聚焦显微镜。 </li><li>使用显微镜激光器TMRM和的Fluo-4的激发和发射光谱的设置。例如，使用543纳米的氦氖和473纳米的氩激光线分别以激发TMRM和的Fluo-4。使用大约5％的低激光功率，以最小化对细胞的任何光损伤。 </li><li>设置显微镜扫描图像每25秒。扫描单元10分钟以建立用于TMRM和的Fluo-4的信号的基线读数。 注:荧光 - 4的信号可能是在静息钙concent微弱或无法检测口粮。 </li><li>加入3微升的40mM的CaCl 2储备溶液直接向细胞中加入300μl的IM成像溶液（1:100稀释），并用移液管轻轻混匀。暂停图像扫描同时加入，如果需要混合的CaCl 2。 注:最终的游离钙离子浓度的离子钙离子浓度在IM成像解决方案可以使用软件进行计算，如Maxchelator WEBMAXC扩展（ <a href="http://www.stanford.edu/%7Ecpatton/maxc.html">http://www.stanford.edu/%7Ecpatton/maxc.html</a> ）或螯合剂11。如何计算最终的游离钙离子离子浓度的详细描述钙离子浓度在下面第6条所述。 </li><li>持续约4分钟图像扫描，然后重复氯化钙增加每4分钟，直到所需的TMRM或荧光- 4信号到达，通常在8到10加法。 </li><li>使用吸管，加入1:100稀释1 mM的FCCP的（决赛Concentration 10μM）直接向细胞消散ΔΨ 微米 。图像细胞再5分钟。实验已经结束。 </li></ol> 5.图像分析<ol><li>一旦成像完成后，确定使用图像分析软件的Fluo-4和TMRM信号的强度，对于具有生物格式的插件（下载&#39;bioformats_package.jar"从http://downloads.openmicroscopy.org/bio例如ImageJ的软件 - 格式/并将其保存在"C: Program Files文件的ImageJ 插件"文件夹）。 </li><li>打开ImageJ的影像文件（例如一个.oif文件）。点击选择工具，并选择感兴趣区域（ROI）的区域包含线粒体。使用初始TMRM信号来选择投资回报率。 </li><li>打开"分析&gt;工具&gt;投资回报率经理"，单击"添加"，以包括强度测量选定的投资回报率。多个ROI可以测量单个图像上进行选择。重复上述步骤，直到所有的投资回报率■找被添加到的ROI管理器。 </li><li>点击"更多&gt;多措施"，确保"衡量所有切片"被选中，那是一排每片"被选中，然后点击"确定"，以获得TMRM和荧光 - 4信号的平均荧光强度的表在每个时间点每个ROI。 </li><li>从所有ROI使用数据来计算用于TMRM和的Fluo-4的信号的平均强度和标准偏差。 </li></ol> 6.计算最终游离钙离子离子浓度钙离子浓度 <ol><li>游离钙离子浓度的[Ca 2+]在IM成像解决方案可以使用软件，如Maxchelator WEBMAXC扩展（待定<a href="http://www.stanford.edu/%7Ecpatton/maxc.html">http://www.stanford.edu/%7Ecpatton/maxc.html</a> ）或螯合剂11。例如，设置WEBMAXC变量EXTENDED如下:温度= 24℃，pH值为7。1，离子强度= 0.162，EGTA = 0.002男，HEDTA = 0.01男， 钙 = 0.0004 M和镁离子 = 0.0078 M.这将导致最终的游离钙离子浓度的离子钙离子浓度 62纳米。请注意，这些程序可以不考虑每一个变量，如试剂的纯度，测量和pH测定精度。用于确定游离Ca 2+浓度的最准确的方法是使用经过校准的钙选择性电极。 </li></ol>

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The authors declare that they have no competing financial interests.

钙起着在许多细胞过程，包括肌肉收缩，神经元信号传导和细胞增殖13的关键作用。在细胞钙离子浓度的增加往往与能源需求有关，与钙能直接刺激线粒体氧化磷酸化，以提高ATP生成3。因此，重要的是，我们必须有效地监测线粒体钙累积，并且能够以比较如何这个功能是由遗传因素和药剂的影响的能力。 该协议中的关键步骤<p class="jove_cont...

线粒体通过充当本地的 Ca 2+缓冲液1在调节细胞内Ca 2+浓度的一个重要的角色。钙离子通过钙单向转运体进入线粒体，通过跨线粒体内膜（ΔΨ 米 ）2存在电化学梯度驱动的方法。一旦线粒体基质内，钙离子可通过刺激三羧酸循环3三限速脱氢酶激活氧化磷酸化。这种刺激保持ΔΨ 男 ，作为正钙离子越过线粒体内膜进入线粒体基质被暂时消散。如果线粒体内的Ca 2+浓度变得非常高，线粒体通透性转换可以启动，导致ΔΨ 米耗散，cessatio氧化磷酸化的n和细胞死亡的信号传导途径4的诱导。 线粒体在细胞中钙的缓冲空间发挥的重要作用，使线粒体钙的准确的监测非常重要。各种方法已被建立并监控线粒体钙，包括使用基于若丹明染料。一个这样的染料，RHOD-2，AM是在分区到线粒体相当有效测量线粒体的 Ca 2+水平5,6。然而，必须注意用作某些染料将在其他细胞器，如脂质体的积累，或保留在细胞胞质溶胶。然而，下游分析可被用来从那些从线粒体7区分这些信号。 监测线粒体钙另一种技术利用荧光记者构造8 </s<html>了&gt;。这些基因编码的探针的好处是，它们可以通过使用内源性N-末端肽，例如人COX VIII亚基的N-末端的定位信号进行具体靶向线粒体。该系统已被用来产生已经证明了线粒体调查钙信号9是非常有用的线粒体靶向探针水母发光蛋白。这些基因编码的探针的主要缺点是，它们需要被引入到由瞬时表达的细胞（其是不适合某些细胞类型可行，并且能够产生不同的结果），或者通过创建稳定表达系统（这是耗时）。 为了规避上述问题，我们已经开发出一种新的协议，同时测量线粒体钙和ΔΨ 米此协议是基于添加了外源钙到透化的细胞一前述方法S ="外部参照"&gt; 10。我们的协议比其他方法三大优势:首先，我们使用荧光- 4，AM和TMRM监测线粒体钙和ΔΨm，即具有非常独特的光谱特性两种染料;其次，将细胞透化，以使的Fluo-4信号仅检测线粒体的Ca 2+和不钙定位于其它细胞器或细胞胞质溶胶;第三，使用的Fluo-4的检测线粒体的 Ca 2+允许快速和简单的细胞染色，否定存在如果使用遗传编码的探针的任何细胞转染或转化的问题。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>ThermoFisher</td>
			<td>10566016</td>
		</tr>
		<tr>
			<td>fetal bovine serum (FBS)</td>
			<td>ThermoFisher</td>
			<td>16000044</td>
		</tr>
		<tr>
			<td>1x phosphate buffered saline (PBS)</td>
			<td>ThermoFisher</td>
			<td>10010023</td>
		</tr>
		<tr>
			<td>100x penicillin/streptomycin (p/s)</td>
			<td>ThermoFisher</td>
			<td>15140122</td>
		</tr>
		<tr>
			<td>0.25% Trypsin / 0.25% EDTA</td>
			<td>ThermoFisher</td>
			<td>25200056</td>
		</tr>
		<tr>
			<td>8-well chambered coverslip</td>
			<td>ibidi</td>
			<td>80826</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>793566</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541&#160;</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma-Aldrich</td>
			<td>746452</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>795488</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270&#160;</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>746495</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375&#160;</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M2670&#160;</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E4378&#160;</td>
		</tr>
		<tr>
			<td>HEDTA</td>
			<td>Sigma-Aldrich</td>
			<td>H8126&#160;</td>
		</tr>
		<tr>
			<td>malate</td>
			<td>Sigma-Aldrich</td>
			<td>M1000</td>
		</tr>
		<tr>
			<td>glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>G1626</td>
		</tr>
		<tr>
			<td>ADP</td>
			<td>Sigma-Aldrich</td>
			<td>A5285</td>
		</tr>
		<tr>
			<td>Ca2+ free Hank&#8217;s buffered salt solution (HBSS)&#160;</td>
			<td>ThermoFisher</td>
			<td>14175-095</td>
		</tr>
		<tr>
			<td>tetramethylrhodamine, methyl ester, perchlorate (TMRM)</td>
			<td>ThermoFisher</td>
			<td>T668</td>
		</tr>
		<tr>
			<td>Verapamil</td>
			<td>Sigma-Aldrich</td>
			<td>V4629</td>
		</tr>
		<tr>
			<td>Fluo-4 acetoxymethyl ester (Fluo-4, AM)</td>
			<td>ThermoFisher</td>
			<td>F14201</td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide (DMSO)</td>
			<td>ThermoFisher</td>
			<td>D12345</td>
		</tr>
		<tr>
			<td>carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP)</td>
			<td>Sigma-Aldrich</td>
			<td>C2920</td>
		</tr>
		<tr>
			<td>digitonin</td>
			<td>Sigma-Aldrich</td>
			<td>D141</td>
		</tr>
		<tr>
			<td>thapsigargin</td>
			<td>Sigma-Aldrich</td>
			<td>T9033</td>
		</tr>
		<tr>
			<td>Pluronic F-127&#160;</td>
			<td>ThermoFisher</td>
			<td>P3000MP&#160;</td>
		</tr>
		<tr>
			<td>hemacytometer</td>
			<td>VWR</td>
			<td>631-0925</td>
		</tr>
		<tr>
			<td>10 cm cell culture dishes</td>
			<td>Corning</td>
			<td>COR430167</td>
		</tr>
		<tr>
			<td>75 cm2 cell culture flasks</td>
			<td>Corning</td>
			<td>COR430641</td>
		</tr>
	</tbody>
</table>

simultaneous measurement of mitochondrial calcium and mitochondrial membrane potential in live cells by fluorescent microscopy

除了在产生的ATP其重要作用，线粒体还充当本地钙（Ca 2+）缓冲器，以紧密地调节细胞内Ca 2+浓度。要做到这一点，线粒体利用电化学势在其内膜（ΔΨ 米 ）隔离的 Ca 2+。 的 Ca 2+的流入线粒体刺激三羧酸循环三个限速脱氢酶，通过氧化磷酸化（OXPHOS）配合增加的电子转移。这种刺激保持ΔΨ 男 ，作为正钙离子越过线粒体内膜进入线粒体基质被暂时消散。 我们在这里描述了使用共聚焦显微镜同时测量线粒体的Ca 2+摄取和ΔΨ 微米活细胞的方法。由透化细胞，线粒体的 Ca 2+能使用荧光钙指示剂的Fluo-4，AM被测定，与使用荧光染料四甲（TMRM）ΔΨ 男 ，甲酯，高氯酸盐测量。本系统的好处是，有荧光染料之间很少光谱重叠，使线粒体的Ca精确测量2+和ΔΨ 米同时。使用顺序除钙等分，线粒体钙的摄取可以被监控，并在其中钙诱导线粒体膜通透性过渡和ΔΨ的损失浓度中号决定。

我们已经使用该协议来检查上143B细胞线粒体的钙12缓冲的增加的能力的MT-ND5突变的影响。在这里所示的例子中，控制143B细胞加载TMRM和的Fluo-4，用毛地黄皂苷透化之前AM。 5分钟成像之后，一个1的8个连续的增补:100稀释的40毫摩尔外源性的CaCl 2被做了，与最终游离Ca 2+离子浓度计算[2+钙。 <p class="jove_content" fo:keep-together.withi...

Watch this Scientific Journal Video about Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy at JoVE.com

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

线粒体可以利用在其内的膜（Δ 膜电势 ）的电化学势螯合钙（Ca 2+），允许它们塑造细胞内的Ca 2+的细胞内信号传导。我们描述了使用荧光染料和共聚焦显微镜同时测量线粒体的Ca 2+摄取和ΔΨ 微米活细胞的方法。

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+ ...

simultaneous-measurement-mitochondrial-calcium-mitochondrial-membrane

Research

JoVE Journal

Biology

18.8K 次观看.  Hudson Institute of Medical Research. 线粒体可以利用在其内的膜（Δ 膜电势 ）的电化学势螯合钙（Ca 2+），允许它们塑造细胞内的Ca 2+的细胞内信号传导。我们描述了使用荧光染料和共聚焦显微镜同时测量线粒体的Ca 2+摄取和ΔΨ 微米活细胞的方法。 

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

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

使用一种基因编码的钙传感器来监测细胞凋亡过程中线粒体钙水平的动态变化

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

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 ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

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 ...

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 ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using short synthetic DNA fragments or plasmids as templates and then extrapolating the results to the whole genome. Alternatively, the number of ribonucleotides present in a genome may be estimated using alkaline gels or Southern blots. More recent in vivo approaches employ Next-generation sequencing allowing genome-wide mapping of ribonucleotides, providing the position and identity of embedded ribonucleotides. However, they do not allow quantitation of the number of ribonucleotides which are incorporated into a genome. Here we describe how to simultaneously map and quantitate the number of ribonucleotides which are incorporated into human mitochondrial DNA in vivo by Next-generation sequencing. We use highly intact DNA and introduce sequence specific double strand breaks by digesting it with an endonuclease, subsequently hydrolyzing incorporated ribonucleotides with alkali. The generated ends are ligated with adapters and these ends are sequenced on a Next-generation sequencing machine. The absolute number of ribonucleotides can be calculated as the number of reads outside the recognition site per average number of reads at the recognition site for the sequence specific endonuclease. This protocol may also be utilized to map and quantitate free nicks in DNA and allows adaption to map other DNA lesions that can be processed to 5&#180;-OH ends or 5&#180;-phosphate ends. Furthermore, this method can be applied to any organism, given that a suitable reference genome is available. This protocol therefore provides an important tool to study DNA replication, 5&#180;-end processing, DNA damage, and DNA repair.

Here we describe a method amenable to simultaneously quantitate and genome-wide map ribonucleotides in highly intact DNA at single-nucleotide resolution, combining enzymatic cleavage of genomic DNA with its alkaline hydrolysis and subsequent 5&#180;-end sequencing.

人线粒体 DNA 中核苷酸的同时定位与定量

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

新鲜分离和内联小鼠脑内皮细胞内钙和膜电位的同时测量

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

Improving the Accuracy of Flow Cytometric Assessment of Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells Through the Inhibition of Efflux Pumps

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic homeostasis have been extensively studied by HSC researchers. Mitochondrial activity levels are reflected in their membrane potentials (&#916;&#936;m), which can be measured by cell-permeant cationic dyes such as TMRM (tetramethylrhodamine, methyl ester). The ability of efflux pumps to extrude these dyes from cells can limit their usefulness, however. The resulting measurement bias is particularly critical when assessing HSCs, as xenobiotic transporters exhibit higher levels of expression and activity in HSCs than in differentiated cells. Here, we describe a protocol utilizing Verapamil, an efflux pump inhibitor, to accurately measure &#916;&#936;m across multiple bone marrow populations. The resulting inhibition of pump activity is shown to increase TMRM intensity in hematopoietic stem and progenitor cells (HSPCs), while leaving it relatively unchanged in mature fractions. This highlights the close attention to dye-efflux activity that is required when &#916;&#936;m-dependent dyes are used, and as written and visualized, this protocol can be used to accurately compare either different populations within the bone marrow, or the same population across different experimental models.

Xenobiotic efflux pumps are highly active in hematopoietic stem and progenitor cells (HSPCs) and cause extrusion of TMRM, a mitochondrial membrane potential fluorescent dye. Here, we present a protocol to accurately measure mitochondrial membrane potential in HSPCs by TMRM in the presence of Verapamil, an efflux pump inhibitor.

通过抑制Efflux泵,提高造血干细胞和造血细胞中线粒体膜电位流细胞测定的准确性

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic ...

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

流细胞测定方法测量造血干细胞和T细胞的线粒体质量及膜电位

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production ...