This work was supported by National Institutes of Health grants GM 100201 to I.F.S, and GM 048071 and GM 065830 to I.P.

我们提出了详细的程序，成像在人类神经母细胞瘤SH-SY5Y细胞局部钙事件。这些程序可适应图像内Ca 2+信号在许多细胞类型8-10。 1.准备细胞<ol><li>培养细胞根据他们的供应商的网站上列出的指令进行成像。 </li><li>在成像之前的几天，收获细胞生长在用1ml的0.25％胰蛋白酶/ EDTA（2-3分钟）的组织培养瓶。下面脱离，加培养基等体积以灭活胰蛋白酶。 </li><li>以每培养皿3-7×10 4个细胞的密度进行使用血球和种子细胞进入玻璃底成像菜细胞计数。选择细胞成像实验，当他们达到60％汇合（2-3天）。 </li></ol> 2.准备解决方案和试剂<ol><li>准备一个缓冲的钙含HEPES盐溶液（ 钙 -HBSS）（MM）组成:135氯化钠，氯化钾5.4，2 氯化钙 ，氯化镁1 2，10 HEPES和10葡萄糖; pH值= 7.4，在室温（RT）下用NaOH。制备的Ca 2+ -HBSS而不在4℃下加入葡萄糖和存储的;添加葡萄糖的溶液在使用前立即。 </li><li>溶解荧光钙离子指 ​​示剂校准- 520（1毫米）的膜透膜物的形式，CI-IP 3（200μM），和EGTA（100毫摩尔）在二甲基亚砜（DMSO）含有20％普流尼克F-127。分装这些试剂进Eppendorf管中并储存，防潮和避光，于-20℃下。 </li></ol> 3.细胞装载膜穿透物加州-520，CI-IP 3和EGTA <ol><li>分别稀释储备溶液膜穿透物校准-520和C的IP 3至5微米和1微米，使最终浓度，对Ca 2+ -HBSS制备"装载缓冲器"。 </li><li>吸立方米lture媒体与钙 -HBSS三次更换介质漂洗细胞。 </li><li>删除钙 -HBSS，并培育细胞样缓冲液在室温下在黑暗中60分钟。 </li><li>卸下装 ​​载的缓冲区，并通过与钙离子-HBSS三次更换介质漂洗细胞。 <ol><li>如果需要的话，在该步骤中，测力传感器用EGTA / AM通过稀释原液至5微米的Ca 2+ -HBSS终浓度，然后孵育细胞在室温在黑暗中30-60分钟。按照此孵育后，进到步骤3.5之前冲洗细胞三次， 钙 -HBSS。 </li></ol></li><li>孵育细胞中的Ca 2+ -HBSS 30分钟，以允许用于脱酯化装载试剂。 </li><li>紧接在成像之前，取代的Ca 2+ -HBSS与"新鲜"的Ca 2+ -HBSS为了除去最后的孵化过程中可能已流入浴任何染料。 </li></ol> 4. 钙 </sup&gt;图像采集<ol><li>将一小滴浸油到100X APO TIRF目标（NA 1.49）。 </li><li>安装在倒置显微镜的阶段成像盘，并使用透射光使细胞进入焦点。它以固定的成像盘，以防止在记录过程中的运动是很重要的。 </li><li>照亮以激发校准-520，并收集用高速EMCCD相机发射的荧光（λ&gt; 510纳米）的细胞用488nm的激光。 注:操作该显微镜软件包允许用户平移一聚焦透镜使激光束被引入或者在目标背孔（用于激发TIRF）以上集中（对于"WF"激发）的末端边缘。 </li><li>使用EMCCD软件，从完整的512×512象素减少，以便收集数据，在必要的时间分辨率来捕捉本地的Ca 2+事件的摄像视场。 注:例如，中心四收购的256×256像素加快并购速度，以66 fps的。另外，也可以使用一个分离的作物模式功能，以进一步增加EMCCD摄像机的帧频为500帧。 </li><li>配置软件自动记录基线活性的几秒钟，递送的UV闪光对细胞和继续录制另一10-30秒。 注:紫外线闪光灯使用通过紫外线反射在显微镜的后部端口分色镜引入氙弧光源递送。紫外线闪光的持续时间和强度被调整凭经验唤起的本地的Ca 2+事件的期望的频率。 </li><li>将文件保存为图像叠加的离线分析。 </li></ol> 5.自动钙图像分析注:可以查看，处理和利用许多商业软件包，分析捕获的数据。但是，我们已经开发了一种算法，以迅速确定自动化的当地的钙信号D分析。该算法中所使用的空间和时间的过滤器的详细说明，生成的ΔF/ f 0的鉴定和/分析例程可以在11找到。该算法已经发展到在开源软件平台的Python运行，并且能够获得，连同样品的实验数据和详细的用户指令，通过电子邮件发送相应的作者（ismith@uci.edu）。 <ol><li>转换文件被导入到自定义编写算法的多平面.tiff格式的文件格式。 </li><li>找到包含分析算法的文件夹，然后双击run.exe。 </li><li>选择.TIFF文件进行分析。 </li><li>选择在打开的对话​​框中分析参数。参见图2A为默认参数的样本数据。 </li><li>确定的黑色电平被从图像堆栈通过移动光标到的视场的，做为n的部分偏移OT包含电池（参见图2B），或通过手动输入黑电平使用"设置黑电平"的提示。 </li><li>观察四个窗口在屏幕上继软件（ 图2C-F）分析。 C是静止从细胞中荧光的单色图像被分析。叠加在该图像的白色方块是事件的地点，判断为装配到事件的二维高斯函数的质心位置。 </li><li>点击每个事件的位置，或者按光标键事件之间循环。在这样的背景中减去，高斯在这些网站更新窗口D的平滑荧光强度的变化（ΔF/女0） 注:红色突出显示的事件，决心从特定网站发起，而不是从&#39;流血通"从活动定位于相邻的站点。较低的蓝色线指示了在所选网站上的荧光信号超过检测的阈值，而黑线识别的事件，从该特定网站的始发（对应于红色突出显示事件）。 </li><li>点击一个红色高亮显示事件更新窗口Ë显示事件的时空演化和窗口F，可以显示该事件的空间分布平均的时间过程，与拟合高斯函数的空间分布在一起。 </li><li>手动检查已确定，这样的文物可以从分析被拒绝的事件。右键单击红色删除这些事件突出事件。 注:在我们的手中， 钙离子通量通过单一IP 3 R声道唤起信号与ΔF/ f 0的约0.11，所以任何检测"事件"明显比这更小的很可能是误报。 </li><li>通过选择"保存到Excel"或导出数据在每个小区所感兴趣的细胞周围绘制，然后选择"保存CellR导出数据17 ;. 注:数据被保存到同一文件夹与原始图像堆栈。 </li></ol>

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The authors declare that they have no competing financial interests.

在这里，我们描述了协议成像培养的哺乳动物细胞局部钙事件使用荧光钙指标。此外，我们描述了一种算法可以自动识别和获取的数据进行分析的直观的用户界面。这里所描述的方法利用荧光钙指标加州-520，但其他许多钙敏感染料如荧光- 3，荧光4，荧光- 8和俄勒冈州绿BAPTA-1进行足够好图像的Ca 2+微区。护理确实需要采取以确保这些指标的高亲和性的版本?...

钙离子（ 钙 ）遍在调节生物学过程，包括基因表达，分泌和在突触可塑性1持久的变化的一个不同的范围。通过这些钙离子可以在这样一个多元化的方式行事的一种方法是通过钙离子的不同的时空格局预示着细胞可以生成。对于胞浆例如全球海拔在平滑肌组织2而较小的[Ca 2+]触发收缩，局部的暂时升高（局部钙微区）刺激基因表达的学习和记忆至关重要的3。 游离胞质内[Ca 2+]被维持在约100纳米处，休息，但能迅速通过钙上升到几个微摩尔以下的Ca 2+的流入到胞质溶胶定位于质膜和由2+ -permeable离子通道钙离子从细胞内的S中解放tores。我们实验室的重点是肌醇1,4,5-三磷酸受体（IP 3 R），其形成位于内质网（ER）膜中的钙释放通道。一旦这两个IP 3和Ca 2+胞浆激活受体位点结合，将IP 3 R通道打开，释放钙离子的ER管腔内隔离。的Ca 2+的释放可保持空间限制到一小簇的IP 3卢比生成的Ca的本地胞质微区2+（钙离子粉扑4），或者，依赖于邻近簇的IP 3 Rs的接近度，可通过钙离子诱导的Ca 2+ -释放（CICR）5,6的过程中多个招聘网站噗传播在整个细胞。 荧光小分子的Ca 2+指示剂染料引入由Roger开发钱学森7，再加上先进的显微镜意马纳克技术，极大地方便了我们的Ca 2+信号的认识。在用于显微镜相机最近的进展，现在允许成像瞬态本地的Ca 2+的事件，如喷以前所未有的空间和时间分辨率。目前可用的EMCCD相机使成像128×128象素，在&gt; 500帧秒-1（fps）的和新一代互补金属氧化物半导体的（CMOS）摄像机提供更高的像素分辨率，并且在稍高的费用，甚至更快的速度噪音水平。在与全内反射（TIRF）显微镜结合使用，现在可以像单钙通道事件8,9。这种方法允许数百同时频道/事件的成像，同时产生大的数据集（每分钟大约 1Gb的），该渲染人工处理，视觉识别和分析不可行和放置一个责任上的自动算法的发展。 钙指标成像在完整的哺乳动物细胞局部钙信号的程序和协议。我们进一步证明了开源环境的Python可以自动识别本地钙事件双方TIRF和传统的宽视场落射荧光（WF）显微成像和分析开发了一种算法。尽管我们描述了这些方法中的IP 3 -生成的Ca 2+信号的情况下，它们是很容易适合于研究在胞浆内[Ca 2+]从各种钙发出位于任一表面2+ -permeable离子通道的局部变化细胞膜或细胞内的细胞器8-10。

<table><tbody><tr><td>Human Neuroblastoma SH-SY5Y cells</td><td>ATCC</td><td>CRL-2266</td><td></td></tr><tr><td>Cal-520/AM</td><td>AAT Bioquest Inc.</td><td>21130</td><td></td></tr><tr><td>ci-IP&lt;sub&gt;3 &lt;/sub&gt;(D-23-&lt;em&gt;O&lt;/em&gt;-Isopropylidene-6-&lt;em&gt;O&lt;/em&gt;-(2-nitro-4,5-dimethoxy)benzyl-&lt;em&gt;myo&lt;/em&gt;-Inositol 1,4,5-trisphosphate-Hexakis(propionoxymethyl) Ester</td><td>SiChem</td><td>cag-iso-2-145-10</td><td></td></tr><tr><td>DMSO/20% pluronic F127</td><td>Invitrogen</td><td>P-3000MP</td><td></td></tr><tr><td>EGTA/AM</td><td>Invitrogen</td><td>E-1219</td><td></td></tr><tr><td>35 mm glass-bottom imaging dishes</td><td>MatTek</td><td>P35G-1.5-14-C</td><td></td></tr></tbody></table>

imaging local ca2+ signals in cultured mammalian cells

胞质的Ca 2+离子的调节在几乎所有类型的细胞的细胞活性的许多方面，控制流程作为广泛作为基因转录，电兴奋性和细胞增殖。 钙离子信号的多样性和特异性的发生在几分钟到局部瞬时钙微区的时间用来生成钙信号采取行动在不同的时间和空间尺度的机制，从细胞级振荡和电磁波派生（ 钙离子喷）持久毫秒。在电子的最新进展乘以CCD（EMCCD）相机，现在允许本地钙信号与128×128像素的空间分辨率成像为&gt; 500帧秒-1（fps）的速率。这种方法是高度并行的，并能够在一个单一实验中同时监测数百个频道或粉扑站点的。然而，所产生的大量数据（ 约 1千兆PER最小值）渲染视觉识别本地钙和分析2+事件不可行。在这里，我们将介绍和演示程序的采集，检测，以及本地IP 3介导的钙信号加载同时使用宽视场落射荧光（WF）和全内反射钙指标完整的哺乳动物细胞分析荧光（TIRF）显微镜。此外，我们描述了开放源码的软件环境的Python，自动化这些地方的钙离子信号的识别和分析中开发了一种算法。该算法本地化的钙释放与子像素分辨率的场所;允许数据的用户查看;输出的荧光信号比时间序列与幅度和动力学数据在Excel兼容的表格。

图1A示出了静息校准-520荧光在人神经母细胞瘤SH-SY5Y细胞还装载了CI-IP 3的WF图像。曝光这些细胞到一个100毫秒的UV闪光灯光释放的i-IP 3引起短暂的Ca 2+喷在离散的位点（由白圈在图1A中说明）。在这些位点测得的荧光的痕迹显示由于知识产权3卢比短暂的开口，随后是慢得多的下降沿相位（ 图1B和1C），为的Ca一个快?...

Watch this Scientific Journal Video about Imaging Local Ca2+ Signals in Cultured Mammalian Cells at JoVE.com

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

成像本地CA<sup&gt; 2+</sup&gt;在培养的哺乳动物细胞信号

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

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Imaging Local Ca2+ Signals in Cultured Mammalian Cells

10.7K 次观看.  University of California, Irvine. Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

视频: Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

在开发皮层网络功能钙成像

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

科研

神经科学

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

C.在体内神经元钙成像技术线虫

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

荧光钙成像及其后续原位杂交，用于异种性前体特征

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

发育生物学

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

前体小鼠膈肌三方突触细胞特异性钙信号的影像学研究

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca2+ signal sources&#8212;&#8220;Ca2+ influx&#8221; from outside the cell and &#8220;Ca2+ release&#8221; from the intracellular Ca2+ store endoplasmic reticulum (ER)&#8212;is considered to underlie the diverse spatio-temporal patterns of Ca2+ signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca2+ imaging method that enables monitoring of the very moment of &#8220;Ca2+ influx&#8221; and &#8220;Ca2+ release&#8221;. OER-GCaMP6f is a genetically encoded Ca2+ indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca2+ release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca2+ signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca2+ indicators described here are, in principle, available for all cell types, even for the in vivo imaging of Caenorhabditis elegans neurons. In this protocol, we introduce Ca2+ imaging in cells from cell lines, neurons, and glial cells in dissociated primary cultures, and describe the preparation of frozen stock of rat cortical neurons.

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Here we present a protocol for Ca2+ imaging in neurons and glial cells, which enables the dissection of Ca2+ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca2+ indicators.

膜靶向 ca 2 + 指标对培养细胞中局部 Ca2 +信号的解剖

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The ...

病毒感染的人肠道类器官单层的钙成像

Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using Genetically Encoded Calcium Indicators

Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory activity, actin dynamics, inflammatory responses, stem cell proliferation, and many other uncharacterized cellular functions. As such, mapping calcium signaling dynamics within the intestinal epithelium can provide insight into homeostatic cellular processes and unveil unique responses to various stimuli. Human intestinal organoids (HIOs) are a high-throughput, human-derived model to study the intestinal epithelium and thus represent a useful system to investigate calcium dynamics. This paper describes a protocol to stably transduce HIOs with genetically encoded calcium indicators (GECIs), perform live fluorescence microscopy, and analyze imaging data to meaningfully characterize calcium signals. As a representative example, 3-dimensional HIOs were transduced with lentivirus to stably express GCaMP6s, a green fluorescent protein-based cytosolic GECI. The engineered HIOs were then dispersed into a single-cell suspension and seeded as monolayers. After differentiation, the HIO monolayers were infected with rotavirus and/or treated with drugs known to stimulate a calcium response. An epifluorescence microscope fitted with a temperature-controlled, humidified live-imaging chamber allowed for long-term imaging of infected or drug-treated monolayers. Following imaging, acquired images were analyzed using the freely available analysis software, ImageJ. Overall, this work establishes an adaptable pipeline for characterizing cellular signaling in HIOs.

作者聚焦：研究肠道上皮信号传导的病毒破坏 – 研究见解和未来方向 

This protocol describes an approach for performing calcium imaging in virus-infected human intestinal organoids and offers an approach to analysis.

使用基因编码的钙指示剂对病毒感染的人肠道类器官单层进行活体钙成像

Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory ...

免疫与感染

Visualizing Shifts on Neuron-Glia Circuit with the Calcium Imaging Technique

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral (dorsal root ganglia) and central tissues (cortex, subventricular zone, organoid) that are dynamically studied in terms of calcium shifts. The model chosen to illustrate the results is the retina, a simple tissue with complex cellular interactions. Calcium is a universal messenger involved in most of the important cellular roles. We explain in a step-by-step protocol how retinal neuron-glial cells in culture can be prepared and evaluated, envisioning calcium shifts. In this model, we differentiate neurons from glia based on their selective response to KCl and ATP. Calcium permeable receptors and channels are selectively expressed in different compartments. To analyze calcium responses, we use ratiometric fluorescent dies such as Fura-2. This probe quantifies free Ca2+ concentration based on Ca2+-free and Ca2+-bound forms, presenting two different peaks, founded on the fluorescence intensity perceived on two wavelengths.

Cell calcium imaging is a versatile methodology to study dynamic signaling of individual cells, on mixed populations in culture or even on awakened animals, based on the expression of calcium-permeable channels/receptors that gives unique functional signatures.

使用钙成像技术可视化神经元 - 神经胶质细胞回路上的位移

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral ...

<meta charset="utf8"></head><body>用于检测 T 细胞中 Ca2+ 微结构域的高分辨率成像技术

Imaging Initial Ca2+ Microdomains in Primary T Cells

Local, sub-second Ca2+ signals, termed Ca2+ microdomains, are highly dynamic and short-lived Ca2+ signals, which result in a global [Ca2+]i elevation and might already determine the fate of a T cell. Upon T cell receptor activation, NAADP is formed rapidly, binding to NAADP binding proteins (HN1L/JPT2, LSM12) and their respective receptors (RyR1, TPC2) sitting on intracellular Ca2+ stores, like the ER and lysosomes, and leading to subsequent release and elevation of [Ca2+]i. To capture these fast and dynamically occurring Ca2+ signals, we developed a high-resolution imaging technique using a combination of two Ca2+ indicators, Fluo-4 AM and FuraRed AM. For postprocessing, an open-source, semi-automated Ca2+ microdomain detection approach was developed based on the programming language Python. Using this workflow, we are able to reliably detect Ca2+ microdomains on a subcellular level in primary murine and human T cells in high temporal and spatial resolution fluorescence videos. This method can also be applied to other cell types, like NK cells and murine neuronal cell lines.

<meta charset="utf8"></head><body>作者聚焦:揭示治疗干预的适应性免疫反应的初始激活

Here, we provide a comprehensive protocol to resolve initial local Ca2+ signals, known as Ca2+ microdomains, in primary murine and human T cells using fluorescence microscopy. This protocol serves as a valuable resource for researchers examining Ca2+ signaling pathways within immune cells and to further unravel their function.

原代 T 细胞中初始 Ca2+ 微结构域的成像

Local, sub-second Ca2+ signals, termed Ca2+ microdomains, are highly dynamic and short-lived Ca2+ signals, which result in a global [Ca2+]i elevation and ...

成像本地CA

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在开发皮层网络功能钙成像

C.在体内神经元钙成像技术线虫

荧光钙成像及其后续原位杂交，用于异种性前体特征

前体小鼠膈肌三方突触细胞特异性钙信号的影像学研究

膜靶向 ca 2 + 指标对培养细胞中局部 Ca^{2 +}信号的解剖

使用基因编码的钙指示剂对病毒感染的人肠道类器官单层进行活体钙成像

使用基因编码的钙指示剂对病毒感染的人肠道类器官单层进行活体钙成像

使用基因编码的钙指示剂对病毒感染的人肠道类器官单层进行活体钙成像

使用钙成像技术可视化神经元 - 神经胶质细胞回路上的位移

原代 T 细胞中初始 Ca²⁺ 微结构域的成像