We express our sincere appreciation for the financial support received from the Connotation Construction Project of the 14th Five-Year Plan of the University of Tibetan Medicine (2022ZYYGH12), the 2022 Open Subjects of the Key Laboratory of Tibetan Medicine and Basic Education of the Ministry of Education at the University of Tibetan Medicine (ZYYJC-22-04), the Key Research and Development Program of Ningxia (2023BEG02012), and the Xinglin Scholar Research Promotion Project of Chengdu University of Traditional Chinese Medicine (XKTD2022013).

The compound solution, protein solution, and dye were introduced into a PBS solution. Subsequently, the samples underwent gradual heating using a digital heating shaking dry bath, and the thermal stability of the proteins was evaluated by measuring the change in fluorescence intensity within the complex system. The detailed steps are outlined below, and Figure 1 illustrates an overview of the protocol.
1. Solution preparation
<ol>
	<li>Prepare a 1 mM umbelliferone solution by adding 0.1 mg of umbelliferone to 616.75 &#181;L of DMSO solution (see Table of Materials).</li>
	<li>Prepare a 200 &#181;g/mL CD40 protein solution by adding 0.1 mg of CD40 to 500 &#181;L of ddH2O solution (see Table of Materials).</li>
	<li>Prepare a 500x orange dye solution by adding 1 &#181;L of 5000x orange dye to 9 &#181;L of phosphate-buffered saline (PBS) (see Table of Materials). 
	NOTE: Perform the above operations on ice, and since the pipette has a minimum range of 0.1 &#181;L, initially dilute the orange protein-dye by a factor of 10 to facilitate subsequent use.</li>
</ol>
2. Dye performance testing
<ol>
	<li>Add the 500x orange dye to a PBS solution and dilute it to achieve dye: PBS ratios of 1:500, 1:1000, 1:2000, and 1:4000. 
	NOTE: Ensure that the final concentration of DMSO does not exceed 2% (v/v).</li>
	<li>Turn on the fluorescent microplate reader and preheat it for 10 min.</li>
	<li>Open the computer and the data acquisition software in sequence (see Table of Materials).</li>
	<li>Click on Instrument, choose the corresponding fluorescence microplate model, and select OK in the data acquisition software. 
	NOTE: Wait for the fluorescence microplate to finish preheating and display the temperature on the panel before opening the data acquisition software to ensure a successful connection between the instrument and the software.</li>
	<li>Click on Acquisition Settings and select Fluorescence in the data acquisition software.</li>
	<li>Edit the program in the Wavelengths interface: Lm1 = 470 nm, 570 nm, and then select OK. 
	NOTE: The orange dye can be excited with UV light at 300 nm or visible light at 470 nm, and emission can be measured at 570 nm.</li>
	<li>Add different concentrations of orange dye (1:500, 1:1000, 1:2000, and 1:4000) and PBS to 96-well plates at 100 &#181;L/well. 
	NOTE: Dissolve the solution in DMSO before adding it to PBS, and vortex it at 2000 x g thoroughly during the preparation process to ensure complete dissolution due to the dye&#39;s tendency to precipitate.</li>
	<li>Place the 96-well plate into the detection stage of the instrument, click on the Read button in the data acquisition software, and measure the fluorescence of each dye concentration 13 times at room temperature, with an interval of 2 min each time.</li>
	<li>Click on the Export button after each determination, select Export to XML XLS TXT, choose All Plates, then select Plate in Output Format, and finally click on OK to save the data in a folder in &#34;XLS&#34; format for further analysis. 
	NOTE: These steps help determine the impact of continuous measurement on the autofluorescence of the orange dye at room temperature and identify the optimal staining concentration.</li>
	<li>Turn on the digital heating shaking dry bath, set the heating temperature to 35 &#176;C, and the heating time to 2 min.</li>
	<li>Prepare the optimal dilution ratio of dye in a 1.5 mL microcentrifuge tube and place it in the digital heating shaking dry bath for 2 min.</li>
	<li>Add PBS solution and the staining solution, heated at 35 &#176;C, to the 96-well plate at a volume of 100 &#181;L per well.</li>
	<li>Place the 96-well plate into the detection stage of the instrument, and click on the Read button in the data acquisition software.</li>
	<li>Set the heating temperature to 40 &#176;C and the heating time to 2 min.</li>
	<li>Pipette the dye solution in the 96-well plate back into the 1.5 mL microcentrifuge tube and heat it in the instrument for 2 min.</li>
	<li>Repeat the operations of steps 2.12-2.15 and step 2.9 to test the absorbance of the dye solution at 35 &#176;C, 40 &#176;C, 45 &#176;C, 50 &#176;C, 55 &#176;C, 60 &#176;C, 65 &#176;C, 70 &#176;C, 75 &#176;C, 80 &#176;C, 85 &#176;C, 90 &#176;C, and 95 &#176;C, respectively. 
	NOTE: These steps help determine the stability of the autofluorescence of the orange dye under continuous heating in a gradient from 35 &#176;C to 95 &#176;C. It is crucial to work swiftly to avoid rapid temperature drops or adding the wrong liquid, which could lead to experimental errors.</li>
</ol>
3. Detection of the temperature melting (Tm) of the protein
<ol>
	<li>Combine CD40 protein and orange dye with PBS solution to achieve final concentrations of 5 &#181;g/mL, 10 &#181;g/mL, 15 &#181;g/mL, 20 &#181;g/mL, and 1:500, respectively.</li>
	<li>Repeat the operations outlined in steps 2.2-2.6 and steps 2.10-2.16 to assess the absorbance of the CD40 protein solution at temperatures of 35 &#176;C, 40 &#176;C, 45 &#176;C, 50 &#176;C, 55 &#176;C, 60 &#176;C, 65 &#176;C, 70 &#176;C, 75 &#176;C, 80 &#176;C, 85 &#176;C, 90 &#176;C, and 95 &#176;C, respectively.</li>
	<li>Reiterate step 2.9 to save and analyze the data, and subsequently calculate the Tm value using data analysis software (see Table of Materials).</li>
	<li>Add CD40 protein, umbelliferone, and orange dye to the PBS solution to obtain final concentrations of 5 &#181;g/mL, 10 &#181;g/mL, 15 &#181;g/mL, 20 &#181;g/mL, 10 &#181;M, and 1:500, respectively. 
	NOTE: Vigorous vortexing during solution preparation is essential to ensure even distribution of the dye, CD40 protein, and umbelliferone in PBS.</li>
	<li>Repeat the operations of steps 3.2-3.3&#160;to measure the absorbance of the CD40-umbelliferone complexes solution at temperatures of 35 &#176;C, 40 &#176;C, 45 &#176;C, 50 &#176;C, 55 &#176;C, 60 &#176;C, 65 &#176;C, 70 &#176;C, 75 &#176;C, 80 &#176;C, 85 &#176;C, 90 &#176;C, and 95 &#176;C, respectively. 
	NOTE: These steps were conducted to determine the Tm values of CD40 protein and CD40-umbelliferone complexes, aiming to identify the optimal CD40 protein binding concentration to umbelliferone.</li>
</ol>

High-Sensitivity Detection of Compound-Protein Binding Using Fluorescence

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The authors have nothing to disclose.

DSF, also known as the thermal shift assay or thermal fluorescence assay, is a technique employed to detect the process of thermal denaturation of proteins in samples by monitoring changes in the fluorescence signal of the test sample or dye during a slow, programmed temperature increase. Initially established by Pantoliano14, DSF serves as a high-throughput method. The main procedure involves elevating the temperature on a computer-controlled heated plate, emitting excitation light using a long-w...

Vicatia thibetica H. Boissieu, a plant in the umbelliferous family, is commonly used in Tibetan medicine and represents one of the essential components of the five basic ingredients (Polygonatum sibiricum Delar. ex Redoute, Asparagus cochinchinensis (Lour.) Merr, Vicatia thibetica H. Boissieu, Oxybaphus himalaicus Edgew., and Gymnadenia conopsea (L.) R. Br.)1. Mainly distributed in southwest China, such as northwest Yunnan, western Sichuan, Tibet, and other areas, its dried root serves as a local substitute for Angelica1. The root, known for its fragrance, is often utilized as a stew seasoning, and the leaves, referred to as Tibetan celery, contribute to delectable dishes. Thus, Vicatia thibetica holds significance not only as a unique medicinal plant but also as a food source in Tibet.
Both domestic and international research indicates that Vicatia thibetica possesses blood-replenishing and qi (power or energy)-invigorating properties, beneficial for regulating menstruation. It is employed to address symptoms of irregular menstrual dysmenorrhea caused by palpitation and blood deficiency, exhibiting pharmacological effects such as antioxidant regulation of body immunity2. The alcohol extract of Vicatia thibetica has demonstrated the ability to restore body mass and organ index in immunocompromised mice induced by cyclophosphamide. Additionally, it increases the activity of superoxide dismutase in serum and reduces the content of malondialdehyde, suggesting an improvement in antioxidant capacity and a balancing effect on lipid peroxidation2,3. Simultaneously, it enhances the number of blood cells and hemoglobin, which not only objectively reflects the body&#39;s hematopoietic function but also plays a crucial role in the immune system2,3.
Umbelliferone, characterized by acicular crystals and a bitter taste, possesses a small molecular weight, is volatile, and can be distilled with steam. It sublimates easily, has low solubility in water, and high solubility in organic matter. As one of the main chemical components of Vicatia thibetica, umbelliferone exhibits immunomodulatory effects on cellular immunity, humoral immunity, and non-specific immunity in hydrocortisone-induced immunosuppression mouse models4,5.
The cell surface molecule CD40, a member of the tumor necrosis factor receptor superfamily, is widely expressed in immune cells6. Its homologous ligand, CD154, also known as CD40L, is a type II transmembrane protein expressed by activated T lymphocytes. CD40 activation has the capacity to up-regulate the expression of co-stimulatory molecules on the surface of dendritic cells (DC) and monocytes. This process promotes the antigen presentation function of major histocompatibility complex (MHC) molecules and further activates CD8+ T cells7.
Macrophages play a crucial role in the formation and regulation of the tumor microenvironment, and CD40 activation can enhance the remodeling of the tumor microenvironment by macrophages8. CD40 signal activation significantly influences the proliferation and activation of B cells. B cells, when activated by CD40, can function as effective antigen-presenting cells. They present antigens, generate effector T cell activity, and thereby contribute to anti-tumor effects9. Moreover, CD40 activation in tumor cells can induce apoptosis and inhibit tumor growth10. CD40 primarily transduces signals by controlling the activity of non-receptor tyrosine protein kinases, including Lyn, Fyn, Syk, and others. Additionally, it has the ability to stimulate Bcl-xL, Cdk4, and Cdk6 proteins, activate Rel/NF-kB transcription factors, and phosphorylate CG-2 and PI3K10.
The Differential Scanning Fluorescence (DSF) method is widely employed to assess the impact of various environmental conditions, such as buffer composition, temperature, and small molecule ligands, on the thermal stability of protein structures. The commonly utilized dye for DSF is an orange, environmentally sensitive, hydrophobic dye. Under normal conditions, the protein structure is folded, concealing its hydrophobic segment internally. As the temperature increases, the protein&#39;s hydrophobic region becomes more exposed, resulting in a gradual breakdown of the natural protein structure. The dye selectively binds to this exposed protein portion, amplifying its fluorescence. Tm values are then calculated by monitoring changes in the fluorescence signal detection11. To a certain extent, variations in Tm values can gauge shifts in protein stability due to mutations, changes in buffers, or ligand binding. Furthermore, it can indicate structural alterations during the protein folding process12. This approach offers precise data, a broad temperature range, high sensitivity, and minimal protein sample loss13.
In this study, fluorescence determination was conducted using a fluorescent microplate reader instead of a fluorescence quantitative polymerase chain reaction (PCR) apparatus. This modification enables DSF detection in laboratories lacking a fluorescent quantitative PCR instrument, making the method less complex and reducing the steps required for instrument setup, thereby simplifying the experimental process. However, there are certain drawbacks to this approach. While the complexity is reduced, the procedure becomes more cumbersome. Manual fluorescence detection at different temperatures is necessary, and automatic and continuous collection of fluorescence from the system cannot be achieved. Thus, this study utilized the DSF technique to explore the interaction between CD40 protein and umbelliferone, providing novel insights into the molecular mechanisms of Tibetan medicine.

<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

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. Umbelliferone, an active ingredient in Tibetan medicine Vicatia thibetica, exhibits an immunomodulatory effect with an unknown mechanism. The CD40 protein is a key target in the immune response. Therefore, this study employs the principle of differential scanning fluorescence technology to analyze the interactions between CD40 protein and umbelliferone using fluorescent enzyme markers. Initially, the stability of the protein fluorescent orange dye was experimentally verified, and the optimal dilution ratio of 1:500 was determined. Subsequently, it was observed that the temperature melting (Tm) value of CD40 protein tended to decrease with an increase in concentration. Interestingly, the interaction between CD40 protein and umbelliferone was found to enhance the thermal stability of CD40 protein. This study represents the first attempt to detect the binding potential of small molecule compounds and proteins using fluorescence microplates and fluorescent dyes. The technique is characterized by high sensitivity and accuracy, promising advancements in the fields of protein stability, protein structure, and protein-ligand interactions, thus facilitating further research and exploration.

The orange dye consistently exhibited stable fluorescence excitation at Ex = 470 nm and Em = 570 nm, both at room temperature and elevated temperatures. An optimal dilution ratio of 1:500 was determined (Figure 2A,B). Detection of the Tm value proved challenging when the concentration of CD40 protein was below 15 &#181;g/mL (Figure 3A,B). However, at a concentration of 15 &#181;g/mL, a stable Tm value of 51.82 &#176;C was detec...

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Author Spotlight: Streamlining PCR Methods for Enhanced Accessibility and Efficiency

This protocol describes a unique method for detecting the binding between compounds and protein molecules, offering the advantages of minimal protein sample loss and high data accuracy.

Detection of CD40 Protein-Umbelliferone Interaction via Differential Scanning Fluorescence

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

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

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393 Views.  University of Tibetan Medicine. This protocol describes a unique method for detecting the binding between compounds and protein molecules, offering the advantages of minimal protein sample loss and high data accuracy.

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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).

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

Biochemistry

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.

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.

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.

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

Immunology and Infection

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.

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

Bioengineering

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.

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

Cancer Research

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.

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.

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.

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

Biology

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).