我们对藏医学院"十四五"规划内涵建设项目（2022ZYYGH12）、藏医药大学教育部藏医与基础教育重点实验室2022年开放课题（ZYYJC-22-04）、宁夏市重点研发计划（2023BEG02012）的资金支持表示衷心感谢。 成都中医药大学杏林学者研究推广项目（XKTD2022013）。

将化合物溶液、蛋白质溶液和染料引入PBS溶液中。随后，使用数字加热振荡干浴对样品进行逐步加热，并通过测量复杂系统内荧光强度的变化来评估蛋白质的热稳定性。下面概述了详细的步骤， 图 1 说明了该协议的概述。
1. 溶液制备
<ol><li>通过将0.1mg伞形酮加入616.75μLDMSO溶液中来制备1mM伞形酮溶液（参见 材料表）。</li>
	<li>通过将 0.1 mg CD40 加入 500 μL ddH2O 溶液中来制备 200 μg/mL CD40 蛋白溶液（参见 材料表）。</li>
	<li>通过将 1 μL 5000x 橙色染料加入 9 μL 磷酸盐缓冲盐水 （PBS） 中来制备 500x 橙色染料溶液（参见 材料表）。 注意:在冰上执行上述操作，由于移液器的最小量程为 0.1 μL，因此最初将橙色蛋白染料稀释 10 倍以方便后续使用。</li>
</ol>2.染料性能检测
<ol><li>将 500x 橙色染料加入 PBS 溶液中并稀释以获得染料:PBS 比例为 1:500、1:1000、1:2000 和 1:4000。 注意:确保 DMSO 的最终浓度不超过 2% （v/v）。</li>
	<li>打开荧光酶标仪并预热10分钟。</li>
	<li>依次打开计算机和数据采集软件（见 材料表）。</li>
	<li>点击 仪器，选择对应的荧光微孔板型号，在数据采集软件中选择 确定 。 注意: 等待荧光微孔板完成预热并在面板上显示温度，然后再打开数据采集软件，以确保仪器与软件成功连接。</li>
	<li>单击 "采集设置 "，然后在数据采集软件中选择" 荧光 "。</li>
	<li>在波长接口中编辑程序:Lm1 = 470 nm、570 nm，然后选择确定。 注:橙色染料可以用 300 nm 的紫外线或 470 nm 的可见光激发，发射可以在 570 nm 处测量。</li>
	<li>将不同浓度的橙色染料（1:500、1:1000、1:2000 和 1:4000）和 PBS 以 100 μL/孔的浓度加入 96 孔板中。 注意:在将溶液添加到PBS之前，将溶液溶解在DMSO中，并在制备过程中以2000× g 彻底涡旋，以确保由于染料的沉淀倾向而完全溶解。</li>
	<li>将96孔板放入仪器的检测台上，点击数据采集软件中的 读取 按钮，在室温下测量每种染料浓度的荧光13次，每次间隔2分钟。</li>
	<li>每次测定后单击"导出"按钮，选择"导出到 XML XLS TXT"，选择"所有板"，然后选择"输出格式的板"，最后单击"确定"以"XLS"格式将数据保存在文件夹中以供进一步分析。 注:这些步骤有助于确定连续测量对室温下橙色染料自发荧光的影响，并确定最佳染色浓度。</li>
	<li>打开数字加热摇动干浴，设置加热温度为35°C，加热时间为2分钟。</li>
	<li>在 1.5 mL 微量离心管中制备染料的最佳稀释比例，并将其置于数字加热振荡干浴中 2 分钟。</li>
	<li>将PBS溶液和在35°C加热的染色溶液以每孔100μL的体积加入96孔板中。</li>
	<li>将 96 孔板放入仪器的检测台，然后单击数据采集软件中的 读取 按钮。</li>
	<li>将加热温度设置为 40 °C，加热时间为 2 分钟。</li>
	<li>将 96 孔板中的染料溶液移回 1.5 mL 微量离心管中，并在仪器中加热 2 分钟。</li>
	<li>重复步骤2.12-2.15和步骤2.9的操作，分别测试染料溶液在35°C、40°C、45°C、50°C、55°C、60°C、65°C、70°C、75°C、80°C、85°C、90°C和95°C下的吸光度。 注:这些步骤有助于确定橙色染料在连续加热下自发荧光的稳定性，梯度为35°C至95°C。 至关重要的是，必须迅速工作，以避免温度快速下降或添加错误的液体，这可能导致实验错误。</li>
</ol>3. 检测蛋白质的温度熔融（Tm）
<ol><li>将 CD40 蛋白和橙色染料与 PBS 溶液混合，最终浓度分别为 5 μg/mL、10 μg/mL、15 μg/mL、20 μg/mL 和 1:500。</li>
	<li>重复步骤 2.2-2.6 和步骤 2.10-2.16 中概述的操作，分别评估 CD40 蛋白溶液在 35 °C、40 °C、45 °C、50 °C、55 °C、60 °C、65 °C、70 °C、75 °C、80 °C、85 °C、90 °C 和 95 °C 的温度下的吸光度。</li>
	<li>重复步骤 2.9 保存和分析数据，然后使用数据分析软件计算 Tm 值（参见 材料表）。</li>
	<li>向 PBS 溶液中加入 CD40 蛋白、伞形酮和橙色染料，分别获得 5 μg/mL、10 μg/mL、15 μg/mL、20 μg/mL、10 μM 和 1:500 的终浓度。 注意:在溶液制备过程中剧烈涡旋对于确保染料、CD40 蛋白和伞形酮在 PBS 中的均匀分布至关重要。</li>
	<li>重复步骤3.2-3.3的操作，分别在35°C，40°C，45°C，50°C，55°C，60°C，65°C，70°C，75°C，80°C，85°C，90°C和95°C的温度下测量CD40-伞形酮复合物溶液的吸光度。 注:执行这些步骤是为了确定 CD40 蛋白和 CD40-伞形酮复合物的 Tm 值，旨在确定与伞形酮的最佳 CD40 蛋白结合浓度。</li>
</ol>

使用荧光高灵敏度检测化合物-蛋白质结合

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作者没有什么可透露的。

DSF，也称为热位移测定或热荧光测定，是一种用于检测样品中蛋白质的热变性过程的技术，通过监测测试样品或染料的荧光信号在缓慢的程序化温度升高期间的变化。DSF 最初由 Pantoliano14 建立，是一种高通量方法。主要步骤包括在计算机控制的加热板上升高温度，使用长波长紫外线灯发射激发光，并通过电荷耦合器件相形成相机捕获荧光染料与蛋白质结合产生的荧光信号强度变?...

Vicatia thibetica H. Boissieu是伞形植物科的一种植物，在藏药中常用，是五种基本成分（Polygonatum sibiricum Delar. ex Redoute， Asparagus cochinchinensis （Lour.）Merr、 Vicatia thibetica H. Boissieu、 Oxybaphus himalaicus Edgew. 和 Gymnadenia conopsea （L.） R. Br.）1.主要分布在我国西南地区，如云南西北部、川西、西藏等地区，其干根作为当归1的局部替代品。这种根以其香味而闻名，经常被用作炖菜调味料，而被称为西藏芹菜的叶子则有助于制作美味的菜肴。因此， 紫 薇不仅作为一种独特的药用植物具有重要意义，而且作为西藏的食物来源也具有重要意义。
国内外研究表明，硫苜蓿具有补血和气（力量或能量）的特性，有利于调节月经。它用于解决由心悸和血虚引起的月经不规则痛经症状，具有抗氧化调节身体免疫力等药理作用2。Vicatia thibetica 的酒精提取物已证明能够恢复环磷酰胺诱导的免疫功能低下小鼠的体重和器官指数。此外，它还增加了血清中超氧化物歧化酶的活性，降低了丙二醛的含量，表明抗氧化能力有所提高，对脂质过氧化有平衡作用2,3。同时，它增强了血细胞和血红蛋白的数量，这不仅客观地反映了人体的造血功能，而且在免疫系统中起着至关重要的作用2,3。
Umbelliferone的特点是针状结晶和苦味，分子量小，易挥发，可以用蒸汽蒸馏。易升华，在水中溶解度低，在有机物中的溶解度高。作为 Vicatia thibetica 的主要化学成分之一，伞形酮在氢化可的松诱导的免疫抑制小鼠模型中对细胞免疫、体液免疫和非特异性免疫具有免疫调节作用 4,5。
细胞表面分子 CD40 是肿瘤坏死因子受体超家族的成员，在免疫细胞中广泛表达6.其同源配体 CD154，也称为 CD40L，是一种由活化的 T 淋巴细胞表达的 II 型跨膜蛋白。CD40 激活具有上调树突状细胞 （DC） 和单核细胞表面共刺激分子表达的能力。该过程促进主要组织相容性复合物 （MHC） 分子的抗原呈递功能，并进一步激活 CD8+ T 细胞7。
巨噬细胞在肿瘤微环境的形成和调控中起着至关重要的作用，CD40的激活可以增强巨噬细胞对肿瘤微环境的重塑8。CD40信号激活显著影响B细胞的增殖和活化。当 B 细胞被 CD40 激活时，可以作为有效的抗原呈递细胞发挥作用。它们呈递抗原，产生效应 T 细胞活性，从而有助于抗肿瘤作用9。此外，肿瘤细胞中的CD40激活可以诱导细胞凋亡并抑制肿瘤生长10。CD40 主要通过控制非受体酪氨酸蛋白激酶的活性来转导信号，包括 Lyn、Fyn、Syk 等。此外，它还具有刺激 Bcl-xL、Cdk4 和 Cdk6 蛋白、激活 Rel/NF-kB 转录因子以及磷酸化 CG-2 和 PI3K10 的能力。
差示扫描荧光 （DSF） 方法被广泛用于评估各种环境条件（如缓冲液组成、温度和小分子配体）对蛋白质结构热稳定性的影响。DSF 常用的染料是一种橙色、环境敏感的疏水染料。在正常情况下，蛋白质结构被折叠，在内部隐藏了其疏水部分。随着温度的升高，蛋白质的疏水区域变得更加暴露，导致天然蛋白质结构逐渐分解。染料选择性地与这种暴露的蛋白质部分结合，放大其荧光。然后通过监测荧光信号检测11的变化来计算Tm值。在一定程度上，Tm 值的变化可以衡量由于突变、缓冲液变化或配体结合导致的蛋白质稳定性变化。此外，它可以指示蛋白质折叠过程中的结构改变12。这种方法提供精确的数据、宽广的温度范围、高灵敏度和最小的蛋白质样品损失13。
在这项研究中，使用荧光酶标仪而不是荧光定量聚合酶链反应 （PCR） 仪器进行荧光测定。这种改进使得在没有荧光定量PCR仪器的实验室中进行DSF检测，使方法不那么复杂，减少了仪器设置所需的步骤，从而简化了实验过程。但是，这种方法存在某些缺点。虽然复杂性降低了，但过程变得更加繁琐。在不同温度下进行手动荧光检测是必要的，并且无法实现从系统中自动连续收集荧光。因此，本研究利用DSF技术探索CD40蛋白与伞形酮之间的相互作用，为藏医分子机制提供了新的见解。

<table><tbody><tr><td>CD40 protein</td><td>MedChemExpress</td><td>HY-P75408</td><td></td></tr><tr><td>DMSO</td><td>Boster Biological Technology Co., Ltd</td><td>PYG0040</td><td></td></tr><tr><td>FlexStation 3 multifunctional microplate reader</td><td>Shanghai Meigu Molecular Instruments Co., LTD</td><td>FlexStation 3</td><td></td></tr><tr><td>OriginPro 8 software</td><td>OriginLab Corporation</td><td>v8.0724(B724)</td><td></td></tr><tr><td>Phosphate buffered saline (1x)</td><td>Gibco</td><td>8120485</td><td></td></tr><tr><td>SoftMax Pro 7.1</td><td>Shanghai Meigu Molecular Instruments Co., LTD</td><td>SoftMax Pro 7.1</td><td></td></tr><tr><td>SSYPRO orange dye</td><td>Sigma</td><td>S5942</td><td></td></tr><tr><td>Umbelliferone</td><td>Shanghai Yuanye Biotechnology Co.</td><td>B21854</td><td></td></tr></tbody></table>

detection of cd40 protein-umbelliferone interaction via differential scanning fluorescence

研究不同分子之间的相互作用是理解疾病发病机制和筛选药物靶点的关键方面。Umbelliferone 是藏药 Vicatia thibetica 中的一种活性成分，具有未知机制的免疫调节作用。CD40蛋白是免疫反应的关键靶标。因此，本研究采用差示扫描荧光技术原理，利用荧光酶标记物分析CD40蛋白与伞形酮之间的相互作用。最初，通过实验验证了蛋白质荧光橙染料的稳定性，并确定了最佳稀释比例为1:500。随后观察到CD40蛋白的温度熔解（Tm）值随着浓度的增加而趋于降低。有趣的是，发现CD40蛋白与伞形酮之间的相互作用增强了CD40蛋白的热稳定性。这项研究代表了使用荧光微孔板和荧光染料检测小分子化合物和蛋白质结合潜力的首次尝试。该技术具有灵敏度和准确度高的特点，在蛋白质稳定性、蛋白质结构和蛋白质-配体相互作用方面取得了可观的进展，从而促进了进一步的研究和探索。

橙色染料在室温和高温下均在 Ex = 470 nm 和 Em = 570 nm 处始终表现出稳定的荧光激发。确定了1:500的最佳稀释比例（图2A，B）。当CD40蛋白浓度低于15μg/ mL时，Tm值的检测被证明是具有挑战性的（图3A，B）。然而，在浓度为15μg/ mL时，可检测到51.82°C的稳定Tm值，随着CD40蛋白浓度的增加，该值降至45.79°C（图3C，D</stro...

Watch this Scientific Journal Video about Author Spotlight: Streamlining PCR Methods for Enhanced Accessibility and Efficiency at JoVE.com

作者聚焦:简化 PCR 方法以提高可及性和效率

该协议描述了一种用于检测化合物和蛋白质分子之间结合的独特方法，具有最小的蛋白质样品损失和高数据准确性的优点。

通过差示扫描荧光检测CD40蛋白-umbellefone相互作用

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. ...

我们的协议提供了一个显着的优势，允许在没有专门设备的情况下使用。与传统的荧光定量PCR方法不同，它简化了实验过程，需要更少的步骤并降低了整体复杂性，使其更安全、更易于进行。这个实验要求操作者对实验程序和荧光酶标仪非常熟悉，不要在溶液加热和荧光检测过程中浪费太多时间。我们将重点介绍伞形酮与 CD14 蛋白结合产生免疫效应的分子机制。

detection-cd40-protein-umbelliferone-interaction-via-differential

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

Detection of CD40 Protein-Umbelliferone Interaction via Differential Scanning Fluorescence

398 次观看.  University of Tibetan Medicine. 我们的协议提供了一个显着的优势，允许在没有专门设备的情况下使用。与传统的荧光定量PCR方法不同，它简化了实验过程，需要更少的步骤并降低了整体复杂性，使其更安全、更易于进行。这个实验要求操作者对实验程序和荧光酶标仪非常熟悉，不要在溶液加热和荧光检测过程中浪费太多时间。我们将重点介绍伞形酮与 CD14 蛋白结合产生免疫效应的分子机制。

视频: 作者聚焦:简化 PCR 方法以提高可及性和效率

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4&#39;,5&#39;-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.

Protein interactions are at the heart of a cell&#39;s function. Calorimetric and spectroscopic techniques are commonly used to characterize them. Here we describe fluorescence anisotropy as a tool to study the interaction between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor-like 1 GTPase (EFL1).

荧光各向异性作为一种工具来研究蛋白质相互作用

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

科研

生物化学

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

低亲和力受体-配体相互作用的细胞外蛋白微阵列技术

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

免疫与感染

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

生物膜荧光探针力:受体 - 配体动力学并行和定量结合诱导细胞内信号作用于单细胞

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

生物工程

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

基于同步成像和流式细胞术检测治疗诱导的衰老癌细胞中的多种荧光衰老标志物

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...

癌症研究

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer composition). The utility of TSA, and specifically of nano-Differential Scanning Fluorimetry (nano-DSF), has been established over the years, both for finding conditions that help stabilize a specific protein and for looking at ligand binding by monitoring changes in the apparent Tm. This paper presents an efficient screening of the Diamond-SGC-iNEXT Poised (DSi-Poised) fragment library (768 compounds) by the use of nano-DSF, monitoring Tm to identify potential fragment binding. The prerequisites regarding protein quality and concentration for performing nano-DSF experiments are briefly outlined followed by a step-by-step protocol that uses a nano-liter robotic dispenser commonly used in structural biology laboratories for preparing the required samples in 96-well plates. The protocol describes how the reagent mixtures are transferred to the capillaries needed for nano-DSF measurements. In addition, this paper provides protocols to measure thermal denaturation (monitoring intrinsic tryptophan fluorescence) and aggregation (monitoring light back-scattering) and the subsequent steps for data transfer and analysis. Finally, screening experiments with three different protein targets are discussed to illustrate the use of this procedure in the context of lead discovery campaigns. The overall principle of the method described can be easily transferred to other fragment libraries or adapted to other instruments.

Monitoring changes in the melting temperature of a target protein (i.e., thermal shift assay, TSA) is an efficient method for screening fragment libraries of a few hundred compounds. We present a TSA protocol implementing robotics-assisted nano-Differential Scanning Fluorimetry (nano-DSF) for monitoring intrinsic tryptophan fluorescence and light back-scattering for fragment screening.

纳米微分扫描荧光仪，用于在基于碎片的铅发现中筛选

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer ...

Integrate Imaging Flow Cytometry and Transcriptomic Profiling to Evaluate Altered Endocytic CD1d Trafficking

Populational analyses of the morphological and functional alteration of endocytic proteins are challenging due to the demand of image capture at a single cell level and statistical image analysis at a populational level. To overcome this difficulty, we used imaging flow cytometry and transcriptomic profiling (RNA-seq) to determine altered subcellular localization of the cluster of differentiation 1d protein (CD1d) associated with impaired endocytic gene expression in human dendritic cells (DCs), which were exposed to the common lipophilic air pollutant benzo[a]pyrene. The colocalization of CD1d and endocytic marker Lamp1 proteins from thousands of cell images captured with imaging flow cytometry was analyzed using IDEAS and ImageJ-Fiji programs. Numerous cellular images with co-stained CD1d and Lamp1 proteins were visualized after gating on CD1d+Lamp1+ DCs using IDEAS. The enhanced CD1d and Lamp1 colocalization upon BaP exposure was further demonstrated using thresholded scatterplots, tested with Mander&#39;s coefficients for co-localized intensity, and plotted based on the percentage of co-localized areas using ImageJ-Fiji. Our data provide an advantageous instrumental and bioinformatic approach to measure protein colocalization at both single and populational cellular levels, supporting an impaired functional outcome of transcriptomic alteration in pollutant-exposed human DCs.

Imaging flow cytometry provides an ideal approach to detect the morphological and functional alteration of cells at individual and populational levels. Disrupted endocytic function for lipid antigen presentation in pollutant-exposed human dendritic cells was demonstrated with a combined transcriptomic profiling of gene expression and morphological demonstration of protein trafficking.

整合影像流式细胞仪和转录组分析, 评估改变的内吞 CD1d 贩运

Populational analyses of the morphological and functional alteration of endocytic proteins are challenging due to the demand of image capture at a single ...

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

蛋白质 - 配体相互作用采用差示扫描荧​​光法测定

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

生物学

A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling contributes to the progression of many diseases. The ligand/receptor pair CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) has raised significant clinical interest, for instance as a potential target for the treatment of cancer and inflammatory diseases. Small molecules as well as therapeutic antibodies that specifically target CXCR4 and inhibit the receptor&#39;s function are therefore considered to be valuable pharmacological tools. Here, a flow cytometry-based cellular assay that allows identification of compounds (e.g., small molecules) that abrogate CXCL12 binding to CXCR4, is described. Essentially, the assay relies on the competition for receptor binding between a fixed amount of fluorescently labeled CXCL12, the natural chemokine agonist for CXCR4, and unlabeled compounds. Hence, the undesirable use of radioactively labeled probes is avoided in this assay. In addition, living cells are used as the source of receptor (CXCR4) instead of cell membrane preparations. This allows easy adaptation of the assay to a plate format, which increases the throughput. This assay has been shown to be a valuable generic drug discovery assay to identify CXCR4-targeting compounds. The protocol can likely be adapted to other GPCRs, at least if fluorescently labeled ligands are available or can be generated. Prior knowledge concerning the intracellular signaling pathways that are induced upon activation of these GPCRs, is not required.

A flow cytometry-based cellular binding assay is described that is primarily used as a screening tool to identify compounds that inhibit the binding of a fluorescently labeled CXC chemokine ligand 12 (CXCL12) to the CXC chemokine receptor 4 (CXCR4).

通过差示扫描荧光检测CD40蛋白-umbellefone相互作用

副本

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此视频中的章节

荧光各向异性作为一种工具来研究蛋白质相互作用

低亲和力受体-配体相互作用的细胞外蛋白微阵列技术

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

生物膜荧光探针力:受体 - 配体动力学并行和定量结合诱导细胞内信号作用于单细胞

基于同步成像和流式细胞术检测治疗诱导的衰老癌细胞中的多种荧光衰老标志物

纳米微分扫描荧光仪，用于在基于碎片的铅发现中筛选

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