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1.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205072

RESUMO

Two-photon microscopy enables monitoring cellular dynamics and communication in complex systems, within a genuine environment, such as living tissues and, even, living organisms. Particularly, its application to understand cellular interactions in the immune system has brought unique insights into pathophysiologic processes in vivo. Simultaneous multiplexed imaging is required to understand the dynamic orchestration of the multiple cellular and non-cellular tissue compartments defining immune responses. Here, we present an improvement of our previously developed method, which allowed us to achieve multiplexed dynamic intravital two-photon imaging, by using a synergistic strategy. This strategy combines a spectrally broad range of fluorophore emissions, a wave-mixing concept for simultaneous excitation of all targeted fluorophores, and an unmixing algorithm based on the calculation of spectral similarities with previously measured fluorophore fingerprints. The improvement of the similarity spectral unmixing algorithm here described is based on dimensionality reduction of the mixing matrix. We demonstrate its superior performance in the correct pixel-based assignment of probes to tissue compartments labeled by single fluorophores with similar spectral fingerprints, as compared to the full-dimensional similarity spectral unmixing approach.


Assuntos
Comunicação Celular/genética , Microambiente Celular/genética , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Algoritmos , Linhagem Celular , Corantes Fluorescentes/química , Fótons
2.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205118

RESUMO

During metastasis, cancer cells that originate from the primary tumor circulate in the bloodstream, extravasate, and form micrometastases at distant locations. Several lines of evidence suggest that specific interactions between cancer cells and endothelial cells, in particular tumor cell adhesion to the endothelium and transendothelial migration, play a crucial role in extravasation. Here we have studied the role of vascular endothelial (VE)-cadherin which is expressed aberrantly by breast cancer cells and might promote such interactions. By comparing different human breast cancer cell lines, we observed that the number of cancer cells that adhered to endothelium correlated with VE-cadherin expression levels. VE-cadherin silencing experiments confirmed that VE-cadherin enhances cancer cell adhesion to endothelial cells. However, in contrast, the number of cancer cells that incorporated into the endothelium was not dependent on VE-cadherin. Thus, it appears that cancer cell adhesion and incorporation are distinct processes that are governed by different molecular mechanisms. When cancer cells incorporated into the endothelial monolayer, they formed VE-cadherin positive contacts with endothelial cells. On the other hand, we also observed tumor cells that had displaced endothelial cells, reflecting either different modes of incorporation, or a temporal sequence where cancer cells first form contact with endothelial cells and then displace them to facilitate transmigration. Taken together, these results show that VE-cadherin promotes the adhesion of breast cancer cells to the endothelium and is involved in the initial phase of incorporation, but not their transmigration. Thus, VE-cadherin might be of relevance for therapeutic strategies aiming at preventing the metastatic spread of breast cancer cells.


Assuntos
Antígenos CD/genética , Neoplasias da Mama/genética , Caderinas/genética , Adesão Celular/genética , Endotélio Vascular/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Técnicas de Cocultura , Endotélio Vascular/patologia , Endotélio Vascular/ultraestrutura , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Imagem Molecular/métodos , Metástase Neoplásica
3.
Nat Commun ; 12(1): 3389, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099672

RESUMO

Bioorthogonal late-stage diversification of amino acids and peptides bears enormous potential for drug discovery and molecular imaging. Despite major accomplishments, these strategies largely rely on traditional, lengthy prefunctionalization methods, heavily involving precious transition-metal catalysis. Herein, we report on a resource-economical manganese(I)-catalyzed C-H fluorescent labeling of structurally complex peptides ensured by direct alkynylation and alkenylation manifolds. This modular strategy sets the stage for unraveling structure-activity relationships between structurally discrete fluorophores towards the rational design of BODIPY fluorogenic probes for real-time analysis of immune cell function.


Assuntos
Técnicas de Química Sintética/métodos , Corantes Fluorescentes/síntese química , Manganês/química , Peptídeos/síntese química , Compostos de Boro/química , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Carbono/química , Catálise , Membrana Celular/metabolismo , Humanos , Hidrogênio/química , Células Jurkat , Microscopia Confocal , Microscopia de Fluorescência , Imagem Molecular/métodos
4.
Methods Mol Biol ; 2268: 207-221, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085271

RESUMO

GPCRs are responsible for activation of numerous downstream effectors. Live cell imaging of these effectors therefore provides a real-time readout of GPCR activity and allows for better understanding of temporal dynamics of GPCR-mediated signaling. Opsins, or optically activatable GPCRs, allow for these signaling pathways to be activated in a spatiotemporally precise and reversible manner. Here, we describe optogenetic methods for activating Gi, Gq, and Gs signaling pathways. Additionally, we present assays for detecting activation of these pathways in real time through live cell imaging of Gßγ translocation, PIP3 increase, PIP2 hydrolysis, cAMP production, and cell migration. These assays can be utilized for GPCR-targeted drug development, as well as for studies of a wide range of GPCR-mediated physiological processes.


Assuntos
Bioensaio/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Imagem Molecular/métodos , Opsinas/metabolismo , Optogenética/métodos , Receptores Acoplados a Proteínas G/metabolismo , Análise de Célula Única/métodos , Movimento Celular/fisiologia , Células Cultivadas , Humanos , Opsinas/genética , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais
5.
Methods Mol Biol ; 2268: 223-232, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085272

RESUMO

Split-TEV assay enables the identification of protein-protein interaction in mammalian cells. This method is based on the split of tobacco etch virus (TEV) protease in two fragments, where each fragment is fused to the candidate proteins predicted to interact. If there is indeed an interaction between both proteins, TEV protease reconstitutes its proteolytic activity and this activity is used to induce the expression of some reporter genes. However, some studies have detected unspecific interaction between membrane proteins due to its higher tendency to aggregate. Here we describe a variation of the Split-TEV method developed with the aim to increase the specificity in the study of G protein-coupled receptor (GPCR) interacting proteins. This approach for monitoring interactions between GPCRs is an easy and robust assay and offers good perspectives in drug discovery.


Assuntos
Bioensaio/métodos , Endopeptidases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Potyvirus/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Cultivadas , Genes Reporter , Humanos , Imagem Molecular/métodos , Potyvirus/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Análise de Célula Única/métodos
6.
Methods Mol Biol ; 2268: 233-248, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085273

RESUMO

Cytosolic ß-arrestins are key regulators of G protein-coupled receptors (GPCRs) by sterically uncoupling G protein activation, facilitating receptor internalization, and/or acting as G protein-independent signaling scaffolds. The current awareness that GPCR ligands may display bias toward G protein signaling or ß-arrestin recruitment makes ß-arrestin recruitment assays important additions to the drug discovery toolbox. This chapter describes two NanoLuc-based methods to monitor ß-arrestin2 recruitment to the human histamine H1 receptor by measuring bioluminescence resonance energy transfer and enzyme-fragment complementation in real-time on living cells with reasonable high throughput. In addition to the detection of agonism, both assay formats can be used to qualitatively evaluate the binding kinetics of antihistamines on the human histamine H1 receptor.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Luciferases/metabolismo , Nanotecnologia/métodos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Histamínicos H1/metabolismo , Análise de Célula Única/métodos , beta-Arrestina 2/metabolismo , Células HEK293 , Humanos , Ligantes , Imagem Molecular/métodos , Ligação Proteica , Transdução de Sinais
7.
Methods Mol Biol ; 2268: 249-274, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085274

RESUMO

An understanding of the kinetic contributions to G protein-coupled receptor pharmacology and signaling is increasingly important in compound profiling. Nonequilibrium conditions are commonly present in vivo, for example, as the drug competes with dynamic changes in hormone or neurotransmitter concentration for the receptor. Under such conditions individual binding kinetic properties of the ligands can influence duration of action, local ligand concentration, and functional properties such as the degree of insurmountable inhibition. Mapping the kinetic patterns of GPCR signaling events elicited by agonists, rather than a peak response at a single timepoint, is often key to predicting their functional impact. This is also a path to a better understanding of the origins of ligand bias, and whether such ligands demonstrate their effects through selection of distinct GPCR conformations, or via their kinetic properties. Recent developments in complementation approaches, based on a small bright shrimp luciferase Nanoluc, provide a new route to kinetic analysis of GPCR signaling in living cells that is amenable to the throughput required for compound profiling. In the NanoBiT luciferase complementation system, GPCRs and effector proteins are tagged with Nanoluc fragments optimized for their low interacting affinity and stability. The interactions brought about by GPCR recruitment of the effector are reproduced by a rapid and reversible increase in NanoBiT luminescence, in the presence of its substrate furimazine. Here we discuss the methods for optimizing and validating the GPCR NanoBiT assays, and protocols for their application to study endpoint and kinetic aspects of agonist and antagonist pharmacology. We also describe how timecourse families of agonist concentration response curves, derived from a single NanoBiT assay experiment, can be used to evaluate the kinetic components in operational model derived parameters of ligand bias.


Assuntos
Bioensaio/métodos , Luciferases/metabolismo , Imagem Molecular/métodos , Receptores Acoplados a Proteínas G/metabolismo , Análise de Célula Única/métodos , Células HEK293 , Humanos , Cinética , Ligantes , Luminescência , Transdução de Sinais
8.
Food Chem ; 362: 130206, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34082289

RESUMO

Legumes are the main sources of folates which are not synthesized in the human body. The five folate species: 5-methyl tetrahydrofolate, tetrahydrofolate, pteroyl glutamate, 5-formyl tetrahydrofolate and 10-formyl tetrahydrofolate were quantitatively determined in legumes seeds and sprouts by a newly developed and validated high performance thin layer chromatography method. High resolution plate imaging hyphenated to mass spectrometry was exploited for fingerprint analysis of tested samples. Results indicated that germination of all seeds resulted in a 2.5-4 fold increase in the content of total folates as well as the individual vitamers. The total amount of folate reached a maximum on the fifth day in the case of black-eyed peas (861 µg/100 g Fresh Weight), white beans (755 µg/100 g FW) and brown lentils (681 µg/100 g FW). 5-CH3-H4 folate was found to be the most dominating folate species reaching its maximum content in day 5 sprouts of black-eyed peas (490 µg/100 g FW).


Assuntos
Cromatografia em Camada Delgada/métodos , Fabaceae/química , Ácido Fólico/análise , Espectrometria de Massas/métodos , Sementes/química , Fabaceae/crescimento & desenvolvimento , Análise de Alimentos/métodos , Análise de Alimentos/estatística & dados numéricos , Germinação , Processamento de Imagem Assistida por Computador , Lens (Planta)/química , Leucovorina/análogos & derivados , Leucovorina/análise , Imagem Molecular/métodos , Análise Multivariada , Reprodutibilidade dos Testes , Sementes/crescimento & desenvolvimento , Tetra-Hidrofolatos/análise
9.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34068875

RESUMO

Atherosclerosis is at the onset of the cardiovascular diseases that are among the leading causes of death worldwide. Currently, high-risk plaques, also called vulnerable atheromatous plaques, remain often undiagnosed until the occurrence of severe complications, such as stroke or myocardial infarction. Molecular imaging agents that target high-risk atheromatous lesions could greatly improve the diagnosis of atherosclerosis by identifying sites of high disease activity. Moreover, a "theranostic approach" that combines molecular imaging agents (for diagnosis) and therapeutic molecules would be of great value for the local management of atheromatous plaques. The aim of this study was to develop and characterize an innovative theranostic tool for atherosclerosis. We engineered oil-in-water nano-emulsions (NEs) loaded with superparamagnetic iron oxide (SPIO) nanoparticles for magnetic resonance imaging (MRI) purposes. Dynamic MRI showed that NE-SPIO nanoparticles decorated with a polyethylene glycol (PEG) layer reduced their liver uptake and extended their half-life. Next, the NE-SPIO-PEG formulation was functionalized with a fully human scFv-Fc antibody (P3) recognizing galectin 3, an atherosclerosis biomarker. The P3-functionalized formulation targeted atheromatous plaques, as demonstrated in an immunohistochemistry analyses of mouse aorta and human artery sections and in an Apoe-/- mouse model of atherosclerosis. Moreover, the formulation was loaded with SPIO nanoparticles and/or alpha-tocopherol to be used as a theranostic tool for atherosclerosis imaging (SPIO) and for delivery of drugs that reduce oxidation (here, alpha-tocopherol) in atheromatous plaques. This study paves the way to non-invasive targeted imaging of atherosclerosis and synergistic therapeutic applications.


Assuntos
Aterosclerose/patologia , Emulsões , Nanopartículas de Magnetita/administração & dosagem , Imagem Molecular/métodos , Anticorpos de Cadeia Única/imunologia , Nanomedicina Teranóstica/métodos , Animais , Aterosclerose/imunologia , Meios de Contraste , Feminino , Humanos , Imageamento por Ressonância Magnética , Nanopartículas de Magnetita/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Polietilenoglicóis
10.
Int J Mol Sci ; 22(11)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34064291

RESUMO

The Arg-Gly-Asp (RGD) peptide shows a high affinity for αvß3 integrin, which is overexpressed in new tumor blood vessels and many types of tumor cells. The radiolabeled RGD peptide has been studied for cancer imaging and radionuclide therapy. We have developed a long-term tumor-targeting peptide DOTA-EB-cRGDfK, which combines a DOTA chelator, a truncated Evans blue dye (EB), a modified linker, and cRGDfK peptide. The aim of this study was to evaluate the potential of indium-111(111In) radiolabeled DOTA-EB-cRGDfK in αvß3 integrin-expressing tumors. The human glioblastoma cell line U-87 MG was used to determine the in vitro binding affinity of the radiolabeled peptide. The in vivo distribution of radiolabeled peptides in U-87 MG xenografts was investigated by biodistribution, nanoSPECT/CT, pharmacokinetic and excretion studies. The in vitro competition assay showed that 111In-DOTA-EB-cRGDfK had a significant binding affinity to U-87 MG cancer cells (IC50 = 71.7 nM). NanoSPECT/CT imaging showed 111In-DOTA-EB-cRGDfK has higher tumor uptake than control peptides (111In-DOTA-cRGDfK and 111In-DOTA-EB), and there is still a clear signal until 72 h after injection. The biodistribution results showed significant tumor accumulation (27.1 ± 2.7% ID/g) and the tumor to non-tumor ratio was 22.85 at 24 h after injection. In addition, the pharmacokinetics results indicated that the 111In-DOTA-EB-cRGDfK peptide has a long-term half-life (T1/2λz = 77.3 h) and that the calculated absorbed dose was safe for humans. We demonstrated that radiolabeled DOTA-EB-cRGDfK may be a promising agent for glioblastoma tumor imaging and has the potential as a theranostic radiopharmaceutical.


Assuntos
Quelantes/metabolismo , Glioblastoma/metabolismo , Oligopeptídeos/metabolismo , Animais , Linhagem Celular Tumoral , Compostos Heterocíclicos com 1 Anel/metabolismo , Xenoenxertos/metabolismo , Humanos , Radioisótopos de Índio/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Imagem Molecular/métodos , Peptídeos Cíclicos/metabolismo , Compostos Radiofarmacêuticos/metabolismo , Ratos , Distribuição Tecidual
11.
Int J Mol Sci ; 22(11)2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34073992

RESUMO

Angiogenesis is an active process, regulating new vessel growth, and is crucial for the survival and growth of tumours next to other complex factors in the tumour microenvironment. We present possible molecular imaging approaches for tumour vascularisation and vitality, focusing on radiopharmaceuticals (tracers). Molecular imaging in general has become an integrated part of cancer therapy, by bringing relevant insights on tumour angiogenic status. After a structured PubMed search, the resulting publication list was screened for oncology related publications in animals and humans, disregarding any cardiovascular findings. The tracers identified can be subdivided into direct targeting of angiogenesis (i.e., vascular endothelial growth factor, laminin, and fibronectin) and indirect targeting (i.e., glucose metabolism, hypoxia, and matrix metallo-proteases, PSMA). Presenting pre-clinical and clinical data of most tracers proposed in the literature, the indirect targeting agents are not 1:1 correlated with angiogenesis factors but do have a strong prognostic power in a clinical setting, while direct targeting agents show most potential and specificity for assessing tumour vascularisation and vitality. Within the direct agents, the combination of multiple targeting tracers into one agent (multimers) seems most promising. This review demonstrates the present clinical applicability of indirect agents, but also the need for more extensive research in the field of direct targeting of angiogenesis in oncology. Although there is currently no direct tracer that can be singled out, the RGD tracer family seems to show the highest potential therefore we expect one of them to enter the clinical routine.


Assuntos
Oncologia/métodos , Imagem Molecular/métodos , Neoplasias/diagnóstico por imagem , Neovascularização Patológica/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Animais , Biomarcadores Tumorais/metabolismo , Hipóxia Celular , Glucose/metabolismo , Humanos , Integrinas/metabolismo , Metaloproteinases da Matriz/metabolismo , Oncologia/instrumentação , Neoplasias/patologia , Neovascularização Patológica/patologia , Oligopeptídeos/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
12.
Nat Commun ; 12(1): 3293, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078903

RESUMO

Dielectric metasurfaces support resonances that are widely explored both for far-field wavefront shaping and for near-field sensing and imaging. Their design explores the interplay between localised and extended resonances, with a typical trade-off between Q-factor and light localisation; high Q-factors are desirable for refractive index sensing while localisation is desirable for imaging resolution. Here, we show that a dielectric metasurface consisting of a nanohole array in amorphous silicon provides a favourable trade-off between these requirements. We have designed and realised the metasurface to support two optical modes both with sharp Fano resonances that exhibit relatively high Q-factors and strong spatial confinement, thereby concurrently optimizing the device for both imaging and biochemical sensing. For the sensing application, we demonstrate a limit of detection (LOD) as low as 1 pg/ml for Immunoglobulin G (IgG); for resonant imaging, we demonstrate a spatial resolution below 1 µm and clearly resolve individual E. coli bacteria. The combined low LOD and high spatial resolution opens new opportunities for extending cellular studies into the realm of microbiology, e.g. for studying antimicrobial susceptibility.


Assuntos
Técnicas Biossensoriais/instrumentação , Espectroscopia Dielétrica/métodos , Imagem Molecular/métodos , Nanoestruturas/química , Silício/química , Análise de Célula Única/métodos , Espectroscopia Dielétrica/instrumentação , Escherichia coli/ultraestrutura , Humanos , Imunoglobulina G/ultraestrutura , Limite de Detecção , Imagem Molecular/instrumentação , Refratometria , Análise de Célula Única/instrumentação , Propriedades de Superfície
13.
Methods Mol Biol ; 2274: 43-51, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050461

RESUMO

The present protocol introduces a new lineage of artificial luciferases (ALucs) with unique optical properties for mammalian cell imaging. The primary candidate sequence was first created with a sequence logo generator, resulting in a total of 11 sibling sequences by extracting consensus amino acids from the alignment of 25 copepod luciferase sequences available in natural luciferase pools in public databases. Phylogenetic analysis shows that the newly fabricated ALucs form an independent branch, genetically isolated from the natural luciferases and from a prior series of ALucs produced by our laboratory using a smaller basis set. The protocol also exemplifies that the new lineage of ALucs was strongly luminescent in living mammalian cells with specific substrate selectivity to native coelenterazine. The success of this approach guides on how to engineer and functionalize marine luciferases for bioluminescence imaging and assays.


Assuntos
Bioensaio/métodos , Luciferases/química , Luciferases/genética , Medições Luminescentes/métodos , Imagem Molecular/métodos , Engenharia de Proteínas/métodos , Animais , Células COS , Chlorocebus aethiops , Copépodes/enzimologia , Luciferases/biossíntese
14.
Methods Mol Biol ; 2274: 89-100, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050465

RESUMO

Advances in live-cell imaging have been accelerated by the development of various fluorescent indicators. However, indicators that are suitable for multicolor imaging remain a challenge to develop. Herein, we have developed a single fluorescent protein (FP)-based indicator using a semirational molecular design and a molecular evolution approach. We first inserted a ligand-binding domain into the vicinity of an FP chromophore to convert the conformational change induced by ligand binding into a change in fluorescence intensity. We then optimized the linker regions between the FP and the ligand-binding domain to greatly expand the dynamic range (F/F0) of the indicator. Our design and optimization methods are highly versatile and can be used to develop any single FP-based indicators, which will further advance the utility of live-cell imaging.


Assuntos
Corantes Fluorescentes/química , Glucose/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Imagem Molecular/métodos , Mutação , Reação em Cadeia da Polimerase/métodos , Evolução Molecular , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Mutagênese Sítio-Dirigida
15.
Methods Mol Biol ; 2274: 103-110, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050466

RESUMO

Coelenterazine (CTZ) is the most general substrate for marine luciferases. The present protocol introduces a near-infrared (NIR ) bioluminescence (BL) imaging of mammalian cells with a cyanine-5 (Cy5) dye-conjugated CTZ . This unique Cy5-conjugated CTZ, named Cy5-CTZ , can act as a dual optical readout emitting both fluorescence (FL) and BL. The Cy5-CTZ exerts through-bond energy transfer (TBET)-based imaging modalities for mammalian cells. This novel derivative, Cy5-CTZ , is intrinsically fluorescent and emits NIR-shifted BL when reacting with an appropriate luciferase , such as Renilla luciferase (RLuc). The protocol exemplifies a unique live-cell imaging with Cy5-CTZ that is optically stable in physiological samples and rapidly permeabilize through plasma membrane and emit NIR-BL in live mammalian cells.


Assuntos
Carbocianinas/química , Imidazóis/química , Luciferases/metabolismo , Substâncias Luminescentes/química , Medições Luminescentes/métodos , Imagem Molecular/métodos , Pirazinas/química , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Células COS , Chlorocebus aethiops , Transferência de Energia
16.
Methods Mol Biol ; 2274: 111-126, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050467

RESUMO

Coelenterazine (CTZ) is a common substrate to most marine luciferases and photoproteins. The present protocol introduces mammalian cell imaging with nine novel dye- and azide-conjugated CTZ analogues, which were synthesized by conjugating a series of fluorescent dyes or an azide group to the C-2 or C-6 position of CTZ backbone. The investigation on the optical properties revealed that azide-conjugated CTZs emit greatly selective bioluminescence (BL) to artificial luciferases (ALucs) and ca. 130 nm blue-shifted BL with Renilla luciferase variant 8.6 (RLuc8.6) in mammalian cells. The corresponding kinetic study explains that azide-conjugated CTZ exerts higher catalytic efficiency than CTZ. Nile red-conjugated CTZ completely showed red-shifted CRET spectra and characteristic BRET spectra with artificial luciferase 16 (ALuc16). The present protocol shows that the minimal spectral overlap occurs among the pairs of [Furimazine/NanoLuc], [6-N3-CTZ/ALuc26], [6-pi-OH-CTZ/RLuc8.6], and [6-N3-CTZ/RLuc8.6] because of the substrate-driven luciferase specificity or color shifts, convincing a cross talk-free multiplex bioassay platform. The present protocol introduces a new toolbox to bioassays and multiplex molecular imaging platforms for mammalian cells.


Assuntos
Azidas/química , Imidazóis/química , Luciferases/metabolismo , Substâncias Luminescentes/química , Medições Luminescentes/métodos , Imagem Molecular/métodos , Pirazinas/química , Animais , Células COS , Chlorocebus aethiops
17.
Methods Mol Biol ; 2274: 217-235, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050475

RESUMO

Recent extensive studies revealed that the intracellular concentration of magnesium ions (Mg2+) is one of the important factors to regulate cellular functions. To evaluate the impact of Mg2+ concentration changes on intracellular signals or events, simultaneous imaging of Mg2+ with those phenomena is a powerful technique. The present protocol describes the synthesis and evaluation of near-infrared (NIR) fluorescent Mg2+-selective probes, named KMG-500 series, and the application to simultaneous imaging of the corresponding intracellular signal transductions and molecular events. The present protocol for multicolor imaging using fluorescent probes in the NIR and visible ranges is highly useful to reveal how multiple molecular events are correlated each other in each single cell.


Assuntos
Trifosfato de Adenosina/metabolismo , Corantes Fluorescentes/química , Hipocampo/metabolismo , Magnésio/metabolismo , Imagem Molecular/métodos , Neurônios/metabolismo , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Células Cultivadas , Potencial da Membrana Mitocondrial , Microscopia de Fluorescência/métodos , Ratos , Transdução de Sinais
18.
Methods Mol Biol ; 2274: 237-243, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050476

RESUMO

Living cells dynamically change their morphology and function according to the cell cycle. Long-term observations of living cells are privileged when we spy the unique, cell cycle-driven molecular events, which cannot be obtained from short-term ones. Mg2+, a metal ion abundant in cells, has been shown to be involved in a variety of physiological phenomena by noninvasive cellular observation using fluorescence microscopy. However, long-term observation of Mg2+ in cells has been a great challenge. Herein, we present a protocol for the long-term microscopic imaging of intracellular Mg2+ levels using a small molecule-protein hybrid fluorescent probe we developed.


Assuntos
Corantes Fluorescentes/química , Magnésio/metabolismo , Imagem Molecular/métodos , Células HEK293 , Humanos , Microscopia de Fluorescência/métodos , Transdução de Sinais
19.
Methods Mol Biol ; 2274: 337-352, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050484

RESUMO

The present protocol introduces a live-cell imaging of secretion activity (LCI-S) that is useful to visualize the real-time release of molecules from individual cells using an immunoassay coupled with total internal reflection fluorescence (FL) microscopy. This novel "live"-cell imaging technique has helped uncover the dynamics of regulated cell "death" by using this new approach. This protocol can observe the final stages of the regulated cell death process via single-cell imaging by targeting the extracellular release of damage-associated molecular patterns (DAMPs) from the cells expressing fluorescence resonance energy transfer (FRET) biosensors, such as a sensor for MLKL activation by RIPK3 based on FRET (SMART) and a sensor for caspase-1 activation based on FRET (SCAT1), which specifically identify the occurrence of regulated cell death processes.


Assuntos
Alarminas/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Imagem Molecular/métodos , Monócitos/patologia , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Morte Celular Regulada , Humanos , Monócitos/metabolismo
20.
Methods Mol Biol ; 2274: 367-384, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050486

RESUMO

Advanced multipurpose cell imaging systems along with integrated rapid quantitation software can enhance and expedite cancer cell culture studies in a variety of applications. Though accurate cell culture studies are an important and necessary component of nearly all cancer biomarker detection and therapy studies, the methods we currently use are of low-throughput, time consuming, and lack accuracy. Hence, it is important to improve several features of the assays to increase the accuracy of their quantitative outputs in most studies. In general, we perform cell culture analysis semimanually by counting a small aliquot of suspended cells using a hemocytometer or viewing a small area of cells on a plate using a bright-field microscope, and then extrapolate the counts or observations to estimate the values for the total numbers of cells. The fundamental problem with this process lies in using techniques, such as extrapolation, which inherently introduces intrasample variability while collecting the cells by enzymatic trypsinization for these assays that are affecting cell growth and other downstream assessments. Fluorescence (FL) microscopy-based assays are also used to image and count cells for various applications, including cell viability, proliferation, apoptosis, cell death, transfection efficiency, protein expression, stem cell properties, colony formation, cytotoxicity, drug dose-response, and treatment efficacy studies. These methods are not optimal for many researches, as they require real-time visualization under a microscope plus manual analysis to determine the final results. Owing to long exposure times for cells under fluorescent light of a microscope, the cells may be exposed to suboptimal conditions that affect cell growth, and with occasional photobleaching of the expressed FL probes. Alternatively, the use of cell imaging systems that integrate both advanced bright-field and FL imaging for cell counting and quantification can be useful. In this protocol, we discuss the advantages of a high-throughput cell imaging system using a whole-plate imaging format when used in various bioimaging studies by highlighting a few applications of the system. The system is designed to fundamentally improve the accuracy and time of cell culture analysis while also allowing us to perform the assay without trypsinization, thus avoiding the need to replicate multiple wells for monitoring cell growth over time.


Assuntos
Apoptose , Proliferação de Células , Ensaios de Triagem em Larga Escala/métodos , Imagem Molecular/métodos , Neoplasias/patologia , Esferoides Celulares/patologia , Técnicas de Cultura de Células , Humanos , Interpretação de Imagem Assistida por Computador , Software , Células Tumorais Cultivadas
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