细胞培养细胞可以种植使用成熟的技术，但必须在1＃玻璃涂上与蜂窝胶粘剂（如聚赖氨酸，polyornithine或层粘连蛋白），以防止细胞分离或在移动成像实验盖玻片镀。 解决方案可以使用各种生理的解决方案，包括细胞培养基钙成像实验。然而，重要的是，以确保该解决方案是无酚红，这大大增加了荧光背景。我们使用Tyrodes解决方案，这是很容易模仿脑脊液，和我们用0.1％的牛血清白蛋白的补充。我们使用60-90毫米氯化钾的去极化，激活电压门控性钙通道和1μM毒胡萝卜素（1 mM的股票在DMSO）或2μm的离子霉素（1 mM的股票在DMSO）激活商店经营的CRAC通道。它往往是方便，从细胞外液表明，钙的海拔是由于钙离子内流中删除的钙。消除钙时，要保持总浓度的二价阳离子（Mg 2 +和钙离子）的恒定。以钠钾时，有必要维持渗透压平衡。 Tyrodes解决方案： <table border="1"><tbody><tr><td></td><td>低血钾2MM的Ca 2 + Tyrodes（毫米） </td><td>低血钾的Ca 2 + Tyrodes（毫米） </td><td>含钾高的2MM的Ca 2 + Tyrodes（毫米） </td><td>含钾高的Ca 2 + Tyrodes（毫米） </td></tr><tr><td>氯化钠</td><td> 129 </td><td> 129 </td><td> 5 </td><td> 5 </td></tr><tr><td>氯化钾</td><td> 5 </td><td> 5 </td><td> 129 </td><td> 129 </td></tr><tr><td>氯化钙</td><td> 2 </td><td> 0 </td><td> 2 </td><td> 0 </td></tr><tr><td>氯化镁</td><td> 1 </td><td> 3 </td><td> 1 </td><td> 3 </td></tr><tr><td>血糖</td><td> 30 </td><td> 30 </td><td> 30 </td><td> 30 </td></tr><tr><td> HEPES </td><td> 25 </td><td> 25 </td><td> 25 </td><td> 25 </td></tr></tbody></table>用NaOH调整pH值至7.4 载入FURA - 2钙染料我们的负载细胞与乙酰氧基甲基酯FURA - 2（FURA - 2 AM），扩散穿过细胞膜和细胞酯酶脱酯化收率FURA - 2游离酸。 FURA - 2装载的确切参数千差万别跨类型的细胞。我们建议测试各种条件准备几个加载解决方案，包含多FURA - 2浓度从1肆虐 - 4微米，在装载解决方案孵化的时间从15分钟到2小时，这和测试在室温下加载各种细胞和在37度。皮层神经元的一个简化的协议如下： <ol><li>首先，准备加入二甲基亚砜50μL到50μg小瓶自Invitrogen FURA - 2上午股市在1毫米。重要的是在氮气包装用干二甲基亚砜，用针头刺破隔垫，以防止水化的二甲基亚砜二甲基亚砜有必要删除。 FURA - 2 AM解决方案后，准备在黑暗干燥的地方。 FURA - 2在DMSO稳定在室温下24小时，是在稳定 - 在几个月的干货集装箱20度。 </li><li>分装2毫升到15毫升锥形管，温暖的37度文化传媒。并添加FURA - 22μLAM股票产生1μMFURA - 2上午的解决方案。旋涡的解决方案1分钟大力。 </li><li>传输装载解决方案为35毫米组织培养皿中，并与细胞转移入菜的盖玻片。 </li><li>孵育30分钟，在一个黑暗的孵化器在37度的神经元。正是时间的孵化。 </li><li>准备好没有FURA - 2 35毫米的菜，含有2 MLS的组织文化传媒。删除加载的解决方案，并在新盘盖玻片。 </li><li>安装成像室盖玻片。从35毫米盘取出盖玻片，并迅速安装到确保防止细胞干燥室。我们使用由华纳仪器制造成像室，包含要安装在底部，安装在顶部形成一个三明治第二盖玻片的细胞，可以让一个10mm盖玻片。两个盖玻片担保室两端允许灌注的解决方案，通过室，真空润滑脂，会议厅和两个管。输入线连接到一个注射器和输出线连接到一个掏空以及连接到真空陷阱吸线。 </li></ol>该显微镜我们使用Eclipse的一个倒置的尼康TE2000 - U显微镜配有一个氙弧灯（萨特仪器），自动化阶段（Ludl），激发滤光片轮（Ludl），和一个冷却的电荷藕合器件（CCD）照相机（滨松Orka二）。显微镜是由一个Macintosh COMPUTER运行开放实验室软件（即兴）。其他几个软件包比例成像。成像，我们用40，60或100倍尼康福陆公司石油浸泡一个NA超过1.2目标。 成像协议<ol><li>校准显微镜阶段。 </li><li>负载Tyrodes照顾，以防止气泡的形成到输入行的解决方案。 </li><li>将腔室灌注线，并通过灌注Tyrodes解决方案再次，照顾，以防止气泡的形成在会议厅内</li><li>客观上放置一个石油的下降，放置在显微镜舞台上腔和集中使用透射光的细胞。 </li><li>检查的使用在340和380纳米，使用目镜照明细胞的荧光。在380应在340昏暗和明亮的休眠细胞。在一般情况下，细胞不应该被照亮超过10或15秒，与紫外线光激发光的强度应与中性密度过滤器，以防止光毒性减少。 </li><li>检查使用相机的细胞，并设置增益和曝光的相机生成的图像是接近饱和（但不饱和）时远低于饱和时，在340 nm照射和照明在380 nm。保持200ms的下面如果有可能的暴露。一旦设置，不改变摄像机的增益或曝光，除非您打算改变背景，RMIN和Rmax（见下文）。 </li><li>收集在每个波长的图像和使用区域的利息率（ROI）的工具来测量在不同的地点，在每个波长的图像背景的强度。 </li><li>平均背景值和背景值进入成像程序的适当位置。背景值将减去在该领域的每一个像素。 </li><li>收集了实践中的比例图像，并调整每个波长的阈值生成的比例图像，其中包括只有细胞，而不是背景，这不是在该地区靠近细胞的边缘嘈杂。 </li><li>设置最低比率值（RMIN）在10％左右，低于最低的比例在该领域的任何单元格。 </li><li>设置RMAX到12倍左右的RMIN。不要更改您计划比较，因为它会比较困难的实验之间的RMIN或RMAX。我们很少改变对我们的成像钻机RMIN和Rmax值。 </li><li>使用自动化的阶段，找到您计划形象的领域，并记录每个字段的位置，使用该软件。在一项实验中，我们一般收取5个领域的观点。自动化的阶段移动到一个领域，收集的比例图像，并移动到下一个字段。 </li><li>设置间隔时间推移，收集0.1和10秒之间除了取决于您期望看到的信号类型的图像。 </li><li>开始实验。 </li><li>钙成像系统测试时，它往往是有益的使用，如高的钾tyrodes（氯化钾65毫米）或离子霉素（2μm的）的刺激，将导致细胞内钙升高和无钙细胞外液（含EGTA和BAPTA钙缓冲区），这将减少钙离子浓度。 </li></ol>分析一旦实验完成后，你将要转换成时间推移钙测量单个细胞或细胞内的感兴趣区域的比例图像。要做到这一点： <ol><li>使用地区的利益工具（ROI）的形象在其中你要衡量钙定义的地区。它通常是有用的，至少有一个投资回报率，涵盖了细胞的细胞体。当确定的投资回报，这是有用的播放图像的影片，以验证该细胞在实验过程中不动。 </li><li>使用该软件来收集时间推移，每幅图像中每个的投资回报率比测量。 </li><li>导入比测量分析程序。您需要查看特定的细胞或投资回报率，平均多个单元格或投​​资回报的钙的痕迹和转换比率测量细胞内钙值。我们使用一套伊戈尔临写，使我们能够做所有这些常见的分析任务，但其他程序如Excel的宏，虽然不太方便，也可以使用。 </li><li>方程[CA] =（RR 分钟 ）/（r 最高 - R）SF * KD可用于转换FURA - 2的比例值，细胞内钙离子浓度。 [CA]是钙离子浓度，R是FURA - 2三百八十○分之三百四比例，RMIN和Rmax中的钙缺乏或饱和浓度的钙存在三百八分之三百四十〇率分别; SF * KD FURA - 2 KD在室温（约120纳米）和定标值的产品。要测量，R R 最小最大和SF * Kd值它在体内或体外校准是有必要执行。需要在体内校准一个结合膜片钳和钙成像，它可以是复杂的，但也很精确。在体外校准可以使用自制的校准室，由薄盖玻片间隔分开的两个生成一层薄薄的解决方案，在其中进行校准的盖玻片组成。作为钙离子浓度的功能的测量FURA - 2的比例值，最方便的方式是使用校准套件包含多个缓冲钙的解决方案和FURA - 2游离酸。这些可从Invitrogen公司（分子探针），并包含使用精确的指示。 </li></ol>

<ol>
	<li>Grynkiewicz, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+generation+of+Ca2++indicators+with+greatly+improved+fluorescence+properties.">A new generation of Ca2+ indicators with greatly improved fluorescence properties.</a> J. Biol. Chem. 260, 3440-3450 (1985).</li><li>Lewis, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitogen-induced+oscillations+of+cytosolic+Ca2++and+transmembrane+Ca2++current+in+human+leukemic+T+cells.">Mitogen-induced oscillations of cytosolic Ca2+ and transmembrane Ca2+ current in human leukemic T cells.</a> Cell Regul. 1, 99-112 (1989).</li><li>Dolmetsch, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+between+intracellular+Ca2++stores+and+depletion-activated+Ca2++channels+generates+[Ca2+]i+oscillations+in+T+lymphocytes.">Signaling between intracellular Ca2+ stores and depletion-activated Ca2+ channels generates [Ca2+]i oscillations in T lymphocytes.</a> J Gen Physiol. 103, 365-388 (1994).</li></ol>

在此演示中，我们已经经历了使用FURA - 2上午进行钙成像的所有步骤。我们作为一个模型的皮层神经元，但可用于实时测量多种细胞的细胞内钙离子钙成像。当执行此过程，重要的是用盖玻片代替塑料，因为塑料是经常在荧光紫外线的波长，这使得成像困难。此外，重要的是预涂层与polyornithine和层粘连蛋白的盖玻片，以防止从分离的细胞。最后，重要的是要记住以避免产生气泡的解决方案，通过成像室时灌流。

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calcium imaging of cortical neurons using fura-2 am

钙成像是一种常见的的技术，用于测量培养细胞中的钙信号。钙成像技术的优势，这是BAPTA为基础的有机分子，Ca2 +离子结合，改变其光谱特性的钙染料。钙染料分为两大类，如FURA - 2和Indo - 1和单波长染料如荧光4的比例度量的染料。比率度量染料改变他们的激发或发射光谱响应钙，使细胞内钙离子浓度在不同的波长的荧光发射或激发的比例确定。使用单波长探测器的比例度量的染料的主要优势是，信噪比是独立的染料浓度，光照强度，和光路长度，使细胞内钙离子的浓度，以确定这些文物的独立。最常见的钙指标之一是FURA - 2，它已经在505 nm处的发射峰从340 nm处的激发峰，380 nm的钙结合。在这里，我们描述了使用FURA - 2，在神经元和其他可兴奋细胞来衡量细胞内钙升高。

Watch this Scientific Journal Video about Calcium Imaging of Cortical Neurons using Fura-2 AM at JoVE.com

Calcium Imaging of Cortical Neurons using Fura-2 AM

钙信号，发挥了关键作用，在许多细胞过程，包括基因表达，存活和分化。在这里，我们将演示如何执行使用FURA - 2的钙成像。钙成像是一种宝贵的的工具来研究细胞内钙离子的实时调控及其信号传导的调控。

使用FURA - 2上午皮层神经元的钙成像

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium ...

calcium-imaging-of-cortical-neurons-using-fura-2-am

Nanyang Technological University

Research

JoVE Journal

Biology

60.7K 次观看.  Stanford University. 钙信号，发挥了关键作用，在许多细胞过程，包括基因表达，存活和分化。在这里，我们将演示如何执行使用FURA - 2的钙成像。钙成像是一种宝贵的的工具来研究细胞内钙离子的实时调控及其信号传导的调控。

视频: Calcium Imaging of Cortical Neurons using Fura-2 AM

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously dissected and prepared. The E18 cortex is, upon dissection, immediately dissociated into individual neurons. It is possible to store E18 cortex in Hibernate E buffer containing B27 at 4&deg;C for up to a week before the dissociation is performed. However, there will be a drop in cell viability. Typically we obtain our E18 Cortex fresh. It is transported to the lab in ice cold Calcium free Magnesium free dissection buffer (CMFM). Upon arrival, trypsin is added to the cortex to a final concentration of 0.125%. The cortex is then incubated at 37&deg;C for 8 minutes. DMEM containing 10% FBS is added to the cortex to stop the reaction. The cortex is then centrifuged at 2500 rpm for 2 minutes. The supernatant is removed and 2 ml of Neural Basal Media (NBM) containing 2% B27 (vol/vol) and 0.25% Glutamax (vol/vol) is added to the cortex which is then re-suspended by pipetting up and down. Next, the cortex is triturated with previously fire polished glass pipettes, each with a successive smaller opening. After triturating, the cortex is once again centrifuged at 2500 rpm for 2 minutes. The supernatant is then removed and the cortex pellet re-suspended with 2 ml of NBM containing B27 and Glutamax. The cell suspension is then passed through a 40 um nylon cell strainer. Next the cells are counted. The neurons are now ready for loading into the neuron microfluidic device.

In this video we demonstrate the preparation of E18 Cortical Rat Neurons.

准备在微流体装置E18条块皮质大鼠神经元

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously ...

科研

生物学

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

在体内2光子层钙成像对小鼠的2 / 3

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

In vivo Imaging of Deep Cortical Layers using a Microprism

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size and have a reflective coating on the hypotenuse to allow internal reflection of excitation and emission light.  The microprism probe simultaneously images multiple cortical layers with a perspective typically seen only in slice preparations.  Images are collected with a large field-of-view (~900 &mu;m).  In addition, we provide details on the non-survival surgical procedure and microscope setup.  Representative results include images of layer V pyramidal neurons from Thy-1 YFP-H mice showing their apical dendrites extending through the superficial cortical layer and extending into tufts.  Resolution was sufficient to image dendritic spines near the soma of layer V neurons.  A tail-vein injection of fluorescent dye reveals the intricate network of blood vessels in the cortex.  Line-scanning of red blood cells (RBCs) flowing through the capillaries reveals RBC velocity and flux rates can be obtained. This novel microprism probe is an elegant, yet powerful new method of visualizing deep cellular structures and cortical function in vivo.

In vivo Imaging of Deep Cortical Layers using a Microprism

Right-angle microprisms inserted into the mouse neocortex allows for deep imaging of multiple cortical layers with a viewpoint typically found in slice. One-millimeter microprisms offer a wide field-of-view (~900 &mu;m) and spatial resolutions sufficient to resolve dendritic spines. We demonstrate layer V neuronal imaging and neocortical vascular imaging using microprisms.

在使用微深皮质层的活体成像

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size ...

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, ...

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 ...

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

直接成像诱导ER钙有针对性的酯酶染料装载（TED）

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

The pancreatic acinar cell is the main parenchymal cell of the exocrine pancreas and plays a primary role in the secretion of pancreatic enzymes into the pancreatic duct. It is also the site for the initiation of pancreatitis. Here we describe how acinar cells are isolated from whole pancreas tissue and intracellular calcium signals are measured. In addition, we describe the techniques of transfecting these cells with adenoviral constructs, and subsequently measuring the leakage of lactate dehydrogenase, a marker of cell injury, during conditions that induce acinar cell injury in vitro. These techniques provide a powerful tool to characterize acinar cell physiology and pathology.

We describe a reproducible method of preparing mouse pancreatic acinar cells from a mouse for the purpose of examining acinar cell calcium signals and cellular injury with physiologically and pathologically relevant stimuli. A method for adenoviral infection of these cells is also provided.

制备胰腺腺泡细胞钙成像的目的，细胞损伤的测量，以及腺病毒感染

The pancreatic acinar  cell is the main parenchymal cell of the exocrine pancreas and plays a primary  role in the secretion of pancreatic enzymes into ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

监测内质网钙稳态使用<em&gt; Gaussia</em&gt;荧光素酶SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

基于FRET钙内波动培养平滑肌细胞使用的肌质网的测量共焦成像

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

Concurrent EEG and Functional MRI Recording and Integration Analysis for Dynamic Cortical Activity Imaging

Electroencephalography (EEG) and functional magnetic resonance imaging (fMRI) are two of the fundamental noninvasive methods for identifying brain activity. Multimodal methods have sought to combine the high temporal resolution of EEG with the spatial precision of fMRI, but the complexity of this approach is currently in need of improvement. The protocol presented here describes the recently developed spatiotemporal fMRI-constrained EEG source imaging method, which seeks to rectify source biases and improve EEG-fMRI source localization through the dynamic recruitment of fMRI sub-regions. The process begins with the collection of multimodal data from concurrent EEG and fMRI scans, the generation of 3D cortical models, and independent EEG and fMRI processing. The processed fMRI activation maps are then split into multiple priors, according to their location and surrounding area. These are taken as priors in a two-level hierarchical Bayesian algorithm for EEG source localization. For each window of interest (defined by the operator), specific segments of the fMRI activation map will be identified as active to optimize a parameter known as model evidence. These will be used as soft constraints on the identified cortical activity, increasing the specificity of the multimodal imaging method by reducing cross-talk and avoiding erroneous activity in other conditionally active fMRI regions. The method generates cortical maps of activity and time-courses, which may be taken as final results, or used as a basis for further analyses (analyses of correlation, causation, etc.) While the method is somewhat limited by its modalities (it will not find EEG-invisible sources), it is broadly compatible with most major processing software, and is suitable for most neuroimaging studies.

An EEG-fMRI multimodal imaging method, known as the spatiotemporal fMRI-constrained EEG source imaging method, is described here. The presented method employs conditionally-active fMRI sub-maps, or priors, to guide EEG source localization in a manner that improves spatial specificity and limits erroneous results.