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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	<li>Berridge, M. J., Lipp, P., Bootman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+versatility+and+universality+of+calcium+signalling.">The versatility and universality of calcium signalling.</a> Nat Rev Mol Cell Biol. 1 (1), 11-21 (2000).</li><li>Hill-Eubanks, D. C., Werner, M. E., Heppner, T. J., Nelson, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling+in+smooth+muscle.">Calcium signaling in smooth muscle.</a> Cold Spring Harb Perspect Biol. 3 (9), a004549 (2011).</li><li>Hagenston, A. 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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 border="0" cellpadding="0" cellspacing="0" width="1055">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:813px;">Name</td>
			<td style="width:124px;">Company</td>
			<td style="width:119px;">Catalogue No.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human Neuroblastoma SH-SY5Y cells</td>
			<td>ATCC</td>
			<td>CRL-2266</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cal-520/AM</td>
			<td>AAT Bioquest Inc.</td>
			<td>21130</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">ci-IP3 (D-23-O-Isopropylidene-6-O-(2-nitro-4.5-dimethoxy)benzyl-myo-Inositol 145-trisphosphate-Hexakis(propionoxymethyl)</td>
			<td>SiChem</td>
			<td>cag-iso-2-145-10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO/20% pluronic F127</td>
			<td>Invitrogen</td>
			<td>P-3000MP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA/AM</td>
			<td>Invitrogen</td>
			<td>E-1219</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 mm glass-bottom imaging dishes</td>
			<td>MatTek</td>
			<td>P35-1.5-14-C</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

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

长期使用宽视场显微镜的成像哺乳动物细胞

科研

生物学

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

技术成像钙 2 +信号在人类精子

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

在哺乳动物细胞的RNAi筛选使命esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

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 ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

在哺乳动物细胞中细胞周期位置分析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

内质网子域的培养细胞中的可视化

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

动态钙的自动分析<sup&gt; 2 +</sup&gt;在图像序列信号

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

中血红素的合成水平在哺乳动物细胞中测量

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.