Name: a semi-automated and reproducible biological-based method to quantify calcium deposition in vitro
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Description: Watch this Scientific Journal Video about A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro at JoVE.com

这项研究由欧盟的地平线2020研究和创新计划资助，根据玛丽·斯克洛多夫斯卡-居里赠款协议第722609和764474，NWO ZonMw（MKMD 40-42600-98-13007）。这项研究得到了BioSPX的支持。WJ-D获得德国研究基金会TRR219项目ID 322900939和项目ID 403041552的资助

1. 细胞接种、维持和钙化诱导
<ol><li>要培养原代细胞，请使用层流气流柜、手套和无菌设备。在进行任何工作之前和之后对手和工作区进行消毒。将所有原代细胞和培养基视为潜在的生物危害，除非另有证明。优选在处置前高压灭菌剩余细胞和培养基。不要化学灭活和高压灭菌，因为这会释放有毒烟雾。</li>
	<li>在未包被的细胞培养板上培养hVSMC。</li>
	<li>在补充有10%-20%FBS和1%Pen / Str...

<ol>
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F., Liberman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifaceted+mechanisms+of+vascular+calcification+in+aging.">Multifaceted mechanisms of vascular calcification in aging.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 39 (7), 1307-1316 (2019).</li><li>Herrmann, J., Babic, M., T&#246;lle, M., vander Giet, M., Schuchardt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+models+for+studying+vascular+calcification.">Research models for studying vascular calcification.</a> International Journal of Molecular Sciences. 21 (6), 2204 (2020).</li><li>Bowler, M. A., Merryman, W. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+automated+procedure+for+the+determination+of+calcium+in+biological+specimens.">An improved automated procedure for the determination of calcium in biological specimens.</a> Analytical Biochemistry. 18 (3), 521-531 (1967).</li><li>Furmanik, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum+stress+mediates+vascular+smooth+muscle+cell+calcification+via+increased+release+of+Grp78+(glucose-regulated+protein,+78+kDa)-loaded+extracellular+vesicles.">Endoplasmic reticulum stress mediates vascular smooth muscle cell calcification via increased release of Grp78 (glucose-regulated protein, 78 kDa)-loaded extracellular vesicles.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 41 (2), 898-914 (2021).</li><li>Jaminon, A. 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在本手稿中，我们描述了一种用于 体外 钙化测定的半自动方法。对于这种方法，应优化hVSMC钙化的三个关键步骤。首先，细胞密度对于hVSMC钙化的发展至关重要。由于缺乏细胞间接触以及在钙化条件下诱导的应激，hVSMC的低密度将导致缓慢或无钙化和细胞死亡21。高细胞密度导致过度融合，之后细胞衰老22 ，钙化发育停止。在孔板中播种大约70%的汇合度至...

血管钙化 （VC） 是心血管疾病发病率和死亡率的独立危险因素1，2，3。长期以来被认为是异位矿物沉积的被动化学过程，现在它似乎是一种可改变的组织愈合反应，涉及各种细胞的积极贡献，包括活化的血管平滑肌细胞（hVSMC）作为疾病的驱动因素4，5。体内VC可以通过多层CT扫描来测量，作为动脉粥样硬化负荷的评估6，7，8。目前，范式转变正在进行中，其中VC严重程度被认为是心血管疾病，II型糖尿病，慢性肾病和衰老的危险因素9，10，11，12，13，14，15。
hVSMC是心血管系统中最丰富的细胞类型，也是VC发展的主要参与者。 体外 hVSMC诱导的钙化是研究心血管疾病的广泛使用的疾病模型16，17。然而，大多数用于体 外 钙化检测的方案使用终点测量，这可能会限制数据采集，需要更多地使用细胞材料，并且可能会减慢研究速度。检测 体外 hVSMC钙化的常用方法包括邻甲酚酞测定法，该测定法测量溶解钙沉积与总蛋白的关系，需要细胞裂解18。此外，使用茜素红染色，其直接与固定细胞或组织上的钙沉积物结合19。要研究邻甲酚酞或茜素红随时间推移的hVSMC钙化，每个时间点需要多次重复，这增加了对生物材料的需求，进而增加了变异性的机会。
在本文中，我们详细介绍了一种新型测定的应用方法，该测定利用hVSMC和荧光成像探针来确定 体外 VC进展，并用作单一终末期钙化测定。我们之前证明，该测定可与邻甲酚酞和茜素红方法直接相媲美，可用于区分不同的培养条件20。除了实时测量外，该测定还可用于确定血清或血浆样品作为临床VC开发的替代标志物的倾向20。这将有助于应用心血管科学和疾病建模的生物学策略。该测定的进一步应用可能是作为转化生物混合系统来评估VC的严重程度或血液成分（如血清或血浆）的进展。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Calcium chloride, 93%, anhydrous</td>
			<td>Thermo Fisher Scientific</td>
			<td>349615000</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 6-well Clear TC-treated well plates</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytation 3 System</td>
			<td>BioTek, Abcoude, The Netherlands</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Merck</td>
			<td>F7524-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetuin-A-Alexa Fluor-546</td>
			<td>Prepared in-house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gen5 Software v3.10</td>
			<td>BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Medium 199</td>
			<td>Thermo Fisher Scientific</td>
			<td>11150059</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydrochloride</td>
			<td>Thermo Fisher Scientific</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10X), pH 7.4</td>
			<td>Thermo Fisher Scientific</td>
			<td>70011044</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>25300062</td>
			<td></td>
		</tr>
	</tbody>
</table>

a semi-automated and reproducible biological-based method to quantify calcium deposition in vitro

血管钙化涉及一系列退行性病变，包括炎症、细胞表型改变、细胞死亡和钙化抑制剂的缺乏，伴随导致血管弹性和功能丧失。血管钙化是许多疾病（包括慢性肾脏病、糖尿病和动脉粥样硬化）发病率和死亡率的重要因素。目前研究血管钙化的研究模型是有限的，仅在体内钙化发展的后期才可行。用于研究血管钙化的体外工具使用终点测量，增加了对生物材料的需求，并有可能将变异性引入研究。我们展示了一种新型荧光标记探针的应用，该探针与人体血管平滑肌细胞的体外钙化发展结合，并确定体外钙化的实时发展。在该协议中，我们描述了我们新开发的钙化测定的应用，这是一种具有潜在转化应用的疾病建模新工具。我们设想该测定法与更广泛的矿物沉积研究相关，包括骨骼、软骨或牙科研究的应用。

结果包括HOECHST染色细胞核的原始图像，RFP标记的钙化和明场图像。可以检测和分析从低（图2）到高（图3）的不同钙化阶段。钙化通常可以使用光学显微镜发现为黑色斑点（图 2D 和图 3B，箭头表示钙化），这对于初步评估和确定何时开始影像学检查很有用。为了提高信噪比，应分析处理后的RFP图像以量化钙...

Watch this Scientific Journal Video about A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro at JoVE.com

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro

心血管疾病是全世界死亡的主要原因。血管钙化大大加重了心血管发病率和死亡率的负担。该协议描述了一种通过荧光成像在 体外 量化血管平滑肌细胞介导的钙沉淀的简单方法。

一种半自动且可重现的基于生物学的体外定量钙沉积的方法

Vascular calcification involves a series of degenerative pathologies, including inflammation, changes to cellular phenotype, cell death, and the absence ...

我们的方法允许使用人血管平滑肌细胞测量患者样本的钙化倾向。这将有助于推动对血管钙化疾病的研究，有助于开发新的治疗方法。该技术使用基于荧光的检测，可提高灵敏度并允许随着时间的推移测量样品，从而减少重复需求并提高稳定性。首先，在37摄氏度的未包被细胞培养瓶上用5%二氧化碳培养人血管平滑肌细胞。在达到70%至90%汇合时分裂细胞。要拆分，请用PBS清洗两次HVSMC。加入胰蛋白酶，在 37 摄氏度下孵育 2 至 5 分钟。通过添加含有血清的培养基来抑制胰蛋白酶反应。将细胞在350 RCF下离心4分钟，并将沉淀重悬于维持培养基中。如图所示制备细胞进行分裂后，将细胞计数并接种到48孔板中以开始钙化实验。在37摄氏度下用5%二氧化碳孵育过夜后，用PBS轻轻洗涤细胞两次，第二次洗涤后小心地吸出所有剩余的PBS。轻轻加入钙化培养基，并在37摄氏度下与5%二氧化碳进一步孵育。每天用光学显微镜检查，直到钙化发生。要通过成像检测钙化，请打开软件并选择协议和新建。选择程序，然后单击设置温度。在弹出窗口中将温度设置为 37 摄氏度，将梯度设置为 1 摄氏度，然后单击确定。从下拉菜单中选择板类型。通过单击图像添加映像步骤。选择反转成像仪，然后单击确定。添加三个成像通道，并将其设置为 DAPI、RFP 和明场。勾选蒙太奇框并设置所需的图像数量和位置。更改重叠以表示每个井的更好覆盖范围。选择要成像的孔。将打开一个新窗口，可以在其中选择感兴趣的井。单击对焦选项以设置对焦模式。将打开一个新窗口。单独设置每个通道。通常，孔自动聚焦在DAPI通道上。将所有其他通道设置为从第一个通道开始固定焦距高度，然后单击“确定”进行设置。关闭窗口。通过单击数据缩减开始预设数据缩减步骤，然后选择图像预处理。通过选择“确定”接受默认设置。通过选择细胞分析设置计数细胞的步骤。从通道下拉菜单中选择 TsF DAPI 图像。在执行成像后设置更多详细信息。通过在弹出窗口标签步骤中选择统计并选择 TsF RFP 作为输入通道来准备 RFP 信号的分析。勾选上限值和下限值框。勾选下方列表中的总面积框。在“颜色效果”列中选择“无”。在弹出窗口中，单击 定制 并勾选 背景 框。按确定。为了获得公平的读数，请按左侧的比率对每个单元格的 RFP 信号进行归一化。在弹出窗口中，选择 RFP 量化总面积。对于下拉菜单中的数据输入 1，然后选择单元格计数作为数据输入 2。最后，选择一个新的数据集名称并按两次确定。如前所述选择“颜色效果”，然后单击左上角 OK.In，选择“文件”并将文件另存为“协议”文件类型。记录第一次钙化发生后每个时间点的观察结果。在启动软件中按立即读取，然后单击现有协议。选择在上一步中创建的协议。程序将提示保存实验。这可以在此阶段完成，也可以稍后通过单击左上角的“保存”按钮手动完成。弹出窗口将要求等待系统加热。打开二氧化碳气体控制器并将其设置为 5%系统达到设定温度后，将出现插入板并读取的提示。按取消。将板从培养箱运输到安全箱中的成像仪，以避免破损和溢出，并符合当地生物安全法规，以便在发生事故时运输活细胞。将板放入读板器中。单击程序。在弹出窗口中，选择预设成像步骤。首先调整 DAPI 通道上的焦点和曝光。取消选中所有通道的自动曝光框，然后单击DAPI通道旁边的显微镜图标。将打开一个新窗口。选择孔以引导焦点。如果信号已经可见，请先点按“自动对焦”，然后点按“自动曝光”。如果没有，请首先增加曝光并重复自动对焦和自动曝光。可以通过选择曝光下拉菜单手动调整曝光。检查多个孔的设置，然后保存设置。以相同的方式调整所有通道的曝光。对于 RFP 通道，请确保从中高钙化的井开始。关闭过程窗口，然后选择顶部任务栏中的绿色“立即阅读”按钮。按照软件指示保存实验。对每个时间点重复此过程。当系统读取印版时，所有图像都在后台自动处理。通过选择要显示的 TsF DAPI 图像来调整细胞计数设置。双击选择所选井。将弹出一个新窗口。选择其中一个图像。在弹出窗口中选择分析选项卡。选择细胞分析细胞计数，然后单击确定。取消选择 RFP 和明场通道。勾选 突出显示对象 框。单击选项以打开菜单。调整大小和强度阈值以包括所有细胞核，但排除碎片，然后单击确定。单击左下角的应用更改以将设置应用于所有图像。同样，选择钙化量中到高的井端图像。要最好地调整信噪比，请单击“任务”菜单中的分析选项卡。选择图像统计信息。取消选择 DAPI 和明场通道。单击选项以打开菜单。勾选阈值异常值框。要设置背景上方信号的非主观阈值，请从任务栏中选择线条工具。将弹出一个窗口。在信号块上画一条线，同时包括背景区域。调整阈值上限和下限值。仅计算背景上方的信号，并排除是否有碎屑或伪影。单击确定并应用更改以将设置传输到所有其他孔。确认在其他孔中准确选择了阈值。设置单元格计数和 RFP 阈值后，导出数据。单击下拉菜单旁边的 Excel 按钮，将数据直接导出到电子表格中。检测并分析从低到高的不同钙化阶段。使用光学显微镜将钙化发现为黑色斑点，这对于初步评估和确定何时开始成像很有用。为了提高信噪比，分析处理后的RFP图像以量化钙化。数据是通过在同一时间点比较两个或多个条件获得的。将数据归一化为细胞计数作为每个细胞的钙化面积。数据也被描述为一个时间序列，显示不同时间点的相同条件。重要的是，数据缩减和分析后的结果仍然反映图像转换和阈值化之前可以看到的内容。

Research

JoVE Journal

Biochemistry

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

科研

生物化学

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

细菌显示文库的半自动化筛选肽亲和试剂的发现与分离结果分析

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

线粒体钙在分离线粒体和培养细胞中的流入分析

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

A Colorimetric Method for Measuring Iron Content in Plants

测定植物铁含量的比色法

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

体外检测 tRNA-Isopentenyl 转移酶活性的实验研究

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

How to Quantify the Fraction of Photoactivated Fluorescent Proteins in Bulk and in Live Cells

如何定量光活化荧光蛋白在大容量和活细胞中的分数

Photoactivatable and -convertible fluorescent proteins (PA-FPs) have been used in fluorescence live-cell microscopy for analyzing the dynamics of cells ...

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

利用高含量成像对增强细胞中的目标参与进行量化

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe ...

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

在 Vivo 钙成像中，在 C. elegans身体壁肌肉

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

一次人气道上皮细胞气道表面液体 pH 值的实时、半自动荧光测量

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) ...

Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore

使用碘电磷诱导红血球中的红血球

Eryptosis, erythrocyte programmed cell death, occurs in a number of hematological diseases and during injury to erythrocytes. A hallmark of eryptotic ...