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1.
Viruses ; 13(8)2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34452373

RESUMO

The development of rapid serological detection methods re urgently needed for determination of neutralizing antibodies in sera. In this study, four rapid methods (ACE2-RBD inhibition assay, S1-IgG detection, RBD-IgG detection, and N-IgG detection) were established and evaluated based on chemiluminescence technology. For the first time, a broadly neutralizing antibody with high affinity was used as a standard for the quantitative detection of SARS-CoV-2 specific neutralizing antibodies in human sera. Sera from COVID-19 convalescent patients (N = 119), vaccinated donors (N = 86), and healthy donors (N = 299) confirmed by microneutralization test (MNT) were used to evaluate the above methods. The result showed that the ACE2-RBD inhibition assay calculated with either ACE2-RBD binding inhibition percentage rate or ACE2-RBD inhibiting antibody concentration were strongly correlated with MNT (r ≥ 0.78, p < 0.0001) and also highly consistent with MNT (Kappa Value ≥ 0.94, p < 0.01). There was also a strong correlation between the two evaluation indices (r ≥ 0.99, p < 0.0001). Meanwhile, S1-IgG and RBD-IgG quantitative detection were also significantly correlated with MNT (r ≥ 0.73, p < 0.0001), and both methods were highly correlated with each other (r ≥ 0.95, p < 0.0001). However, the concentration of N-IgG antibodies showed a lower correlation with the MNT results (r < 0.49, p < 0.0001). The diagnostic assays presented here could be used for the evaluation of SARS-CoV-2 vaccine immunization effect and serological diagnosis of COVID-19 patients, and could also have guiding significance for establishing other rapid serological methods to surrogate neutralization tests for SARS-CoV-2.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Teste Sorológico para COVID-19/métodos , Vacinas contra COVID-19/imunologia , COVID-19/virologia , Imunoensaio/métodos , Medições Luminescentes/métodos , SARS-CoV-2/imunologia , COVID-19/sangue , COVID-19/imunologia , COVID-19/prevenção & controle , Teste Sorológico para COVID-19/instrumentação , Vacinas contra COVID-19/administração & dosagem , Humanos , SARS-CoV-2/genética , Vacinação
2.
Molecules ; 26(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361800

RESUMO

Thin-layer chromatography (TLC) bioautography is an evolving technology that integrates the separation and analysis technology of TLC with biological activity detection technology, which has shown a steep rise in popularity over the past few decades. It connects TLC with convenient, economic and intuitive features and bioautography with high levels of sensitivity and specificity. In this study, we discuss the research progress of TLC bioautography and then establish a definite timeline to introduce it. This review summarizes known TLC bioautography types and practical applications for determining antibacterial, antifungal, antitumor and antioxidant compounds and for inhibiting glucosidase, pancreatic lipase, tyrosinase and cholinesterase activity constitutes. Nowadays, especially during the COVID-19 pandemic, it is important to identify original, natural products with anti-COVID potential compounds from Chinese traditional medicine and natural medicinal plants. We also give an account of detection techniques, including in situ and ex situ techniques; even in situ ion sources represent a major reform. Considering the current technical innovations, we propose that the technology will make more progress in TLC plates with higher separation and detection technology with a more portable and extensive scope of application. We believe this technology will be diffusely applied in medicine, biology, agriculture, animal husbandry, garden forestry, environmental management and other fields in the future.


Assuntos
Cromatografia em Camada Delgada/métodos , Descoberta de Drogas/métodos , Medições Luminescentes/métodos , Animais , Anti-Infecciosos/isolamento & purificação , Antineoplásicos/isolamento & purificação , Antioxidantes/isolamento & purificação , Inibidores Enzimáticos/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana/métodos , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Plantas Medicinais/química , Sensibilidade e Especificidade
3.
Molecules ; 26(14)2021 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-34299498

RESUMO

Among all bioluminescent organisms, the firefly is the most famous, with a high luminescent efficiency of 41%, which is widely used in the fields of biotechnology, biomedicine and so on. The entire bioluminescence (BL) process involves a series of complicated in-vivo chemical reactions. The BL is initiated by the enzymatic oxidation of luciferin (LH2). However, the mechanism of the efficient spin-forbidden oxygenation is far from being totally understood. Via MD simulation and QM/MM calculations, this article describes the complete process of oxygenation in real protein. The oxygenation of luciferin is initiated by a single electron transfer from the trivalent anionic LH2 (L3-) to O2 to form 1[L•2-…O2•-]; the entire reaction is carried out along the ground-state potential energy surface to produce the dioxetanone (FDO-) via three transition states and two intermediates. The low energy barriers of the oxygenation reaction and biradical annihilation involved in the reaction explain this spin-forbidden reaction with high efficiency. This study is helpful for understanding the BL initiation of fireflies and the other oxygen-dependent bioluminescent organisms.


Assuntos
Vaga-Lumes/metabolismo , Luciferases de Vaga-Lume/metabolismo , Substâncias Luminescentes/metabolismo , Animais , Compostos Heterocíclicos com 1 Anel/metabolismo , Luminescência , Medições Luminescentes/métodos , Oxirredução
5.
Methods Mol Biol ; 2350: 229-237, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331288

RESUMO

The recent development of the bright luciferase NanoLuc (Nluc) has greatly improved the sensitivity of bioluminescence imaging, enabling real-time cellular imaging with high spatial resolution. However, the limited color variants of Nluc have restricted its wider application to multicolor imaging of biological phenomena. To address this issue, we developed five new spectral variants of the bright bioluminescent protein with emissions across the visible spectrum. In this chapter, we describe the following two protocols for single-cell bioluminescence imaging: (a) multicolor bioluminescence imaging of subcellular structures and (b) multicolor calcium imaging in single living cells.


Assuntos
Cálcio/metabolismo , Medições Luminescentes/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Imagem Molecular/métodos , Análise de Célula Única/métodos , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Linhagem Celular , Expressão Gênica , Genes Reporter , Humanos
6.
Methods Mol Biol ; 2328: 253-259, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34251631

RESUMO

Enhancers are one of the main classes of cis-regulatory elements (CREs) in the regulation of plant gene expression. Plant enhancers can be predicted based on genomic signatures associated with open chromatin. However, predicted enhancers need to be validated experimentally. We developed an experimental system for rapid enhancer validation. Predicted enhancer candidates are cloned into a vector containing a minimal 35S promoter and a luciferase reporter gene. The construct is then agroinfiltrated into Nicotiana benthamiana leaves followed by bioluminescence signal detection and analysis. Positive bioluminescence signals indicate the enhancer function of each candidate, and the relative signal strength from different enhancers can be quantitatively measured and compared. In summary, we have developed an efficient and rapid plant enhancer validation assay based on a bioluminescent luciferase reporter and agroinfiltration-based N. benthamiana leaf transient expression. This assay can be used for the initial screening of candidate enhancers that are active in leaf tissue. The system can potentially be used to examine the activity of candidate enhancers under different environmental conditions.


Assuntos
Elementos Facilitadores Genéticos , Genes Reporter , Medições Luminescentes/métodos , Tabaco/metabolismo , Agrobacterium/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Vetores Genéticos , Luciferases/genética , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação , Imagem com Lapso de Tempo , Tabaco/genética , Tabaco/crescimento & desenvolvimento , Transformação Genética
7.
J Immunol ; 207(4): 1211-1221, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34312257

RESUMO

Long half-life of therapeutic Abs and Fc fusion proteins is crucial to their efficacy and is, in part, regulated by their interaction with neonatal Fc receptor (FcRn). However, the current methods (e.g., surface plasmon resonance and biolayer interferometry) for measurement of interaction between IgG and FcRn (IgG/FcRn) require either FcRn or IgG to be immobilized on the surface, which is known to introduce experimental artifacts and have led to conflicting data. To study IgG/FcRn interactions in solution, without a need for surface immobilization, we developed a novel (to our knowledge), solution-based homogeneous binding immunoassay based on NanoBiT luminescent protein complementation technology. We optimized the assay (NanoBiT FcRn assay) for human FcRn, mouse FcRn, rat FcRn, and cynomolgus FcRn and used them to determine the binding affinities of a panel of eight Abs. Assays could successfully capture the modulation in IgG/FcRn binding based on changes in Fc fragment of the Abs. We also looked at the individual contribution of Fc and F(ab)2 on the IgG/FcRn interaction and found that Fc is the main driver for the interaction at pH 6. Our work highlights the importance of using orthogonal methods to validate affinity data generated using biosensor platforms. Moreover, the simple add-and-read format of the NanoBiT FcRn assay is amenable for high-throughput screening during early Ab discovery phase.


Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Imunoensaio/métodos , Medições Luminescentes/métodos , Receptores Fc/imunologia , Sequência de Aminoácidos , Animais , Técnicas Biossensoriais/métodos , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Testes Imunológicos/métodos , Camundongos , Ligação Proteica/imunologia , Ratos
8.
Methods Mol Biol ; 2277: 175-185, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080152

RESUMO

The Protoporphyrin IX-Triplet State Lifetime Technique (PpIX-TSLT) has been proposed by us as a potential clinical noninvasive tool for monitoring mitochondrial function. We have been working on the development of mitochondrial respirometry for monitoring mitochondrial oxygen tension (mitoPO2) and mitochondrial oxygen consumption (mitoVO2) in skin. In this work, we describe the principles of the method in small experimental animals.


Assuntos
Mitocôndrias/metabolismo , Consumo de Oxigênio , Ácido Aminolevulínico/farmacologia , Animais , Temperatura Corporal , Desenho de Equipamento , Medições Luminescentes/instrumentação , Medições Luminescentes/métodos , Protoporfirinas/química , Ratos Wistar , Respiração Artificial , Pele/efeitos dos fármacos , Traqueotomia
9.
Cancer Sci ; 112(9): 3796-3809, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34145937

RESUMO

Tissue-clearing technology is an emerging imaging technique currently utilized not only in neuroscience research but also in cancer research. In our previous reports, tissue-clearing methods were used for the detection of metastatic tumors. Here, we showed that the cell cycles of primary and metastatic tumors were visualized by tissue-clearing methods using a reporter system. First, we established cancer cell lines stably expressing fluorescent ubiquitination-based cell cycle indicator (Fucci) reporter with widely used cancer cell lines A549 and 4T1. Fluorescence patterns of the Fucci reporter were investigated in various tumor inoculation models in mice. Interestingly, fluorescence patterns of the Fucci reporter of tumor colonies were different between various organs, and even among colonies in the same organs. The effects of antitumor drugs were also evaluated using these Fucci reporter cells. Of the three antitumor drugs studied, 5-fluorouracil treatment on 4T1-Fucci cells resulted in characteristic fluorescent patterns by the induction of G2 /M arrest both in vitro and in vivo. Thus, the combination of a tissue-clearing method with the Fucci reporter is useful for analyzing the mechanisms of cancer metastasis and drug resistance.


Assuntos
Adenocarcinoma de Pulmão/patologia , Neoplasias da Mama/patologia , Ciclo Celular , Medições Luminescentes/métodos , Neoplasias Pulmonares/patologia , Células A549 , Animais , Antimetabólitos Antineoplásicos/administração & dosagem , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , Feminino , Fluoruracila/administração & dosagem , Genes Reporter , Vetores Genéticos/genética , Humanos , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia de Fluorescência/métodos , Transfecção , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Cancer Sci ; 112(9): 3484-3490, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34187084

RESUMO

For successful immunotherapy for cancer, it is important to understand the immunological status of tumor antigen-specific CD8+ T cells in the tumor microenvironment during tumor progression. In this study, we monitored the behavior of B16OVA-Luc cells in mice immunized with a model tumor antigen ovalbumin (OVA). Using bioluminescence imaging, we identified the time series of OVA-specific CD8+ T-cell responses during tumor progression: initial progression, immune control, and the escape phase. As a result of analyzing the status of tumor antigen-specific CD8+ cells in those 3 different phases, we found that the expression of NKG2D defines tumor-reacting effector CD8+ T cells. NKG2D may control the fate and TOX expression of tumor-reacting CD8+ T cells, considering that NKG2D blockade in OVA-vaccinated mice delayed the growth of the B16OVA-Luc2 tumor and increased the presence of tumor-infiltrating OVA-specific CD8+ T cells.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Microambiente Tumoral/imunologia , Animais , Antígenos de Neoplasias/administração & dosagem , Antígenos de Neoplasias/metabolismo , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/farmacocinética , Bromodesoxiuridina/administração & dosagem , Bromodesoxiuridina/farmacocinética , Interferon gama/deficiência , Interferon gama/genética , Luciferases/metabolismo , Medições Luminescentes/métodos , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/administração & dosagem , Ovalbumina/metabolismo , Neoplasias Cutâneas/patologia , Vacinação/métodos
11.
Anal Bioanal Chem ; 413(17): 4407-4416, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34081166

RESUMO

A novel copper-based metal-organic framework (Cu-MOF) with a large specific surface area and high porosity was synthesized. The Cu-MOF was a good peroxidase-mimicking enzyme and showed a high affinity with hydrogen peroxide in a wide pH range. The catalytic mechanism of Cu-MOF has been studied further based on comparing the characteristic of the Cu-MOF with some isomorphic MOFs. The catalytic activity center of Cu-MOF was determined to be the cupric ion rather than the ligand, which effectively promoted the generation of free radicals and electron transfer in the reaction progress. The high affinity of Cu-MOF to hydrogen peroxide proved it as an ideal catalyst for the chemiluminescence (CL) reaction involving hydrogen peroxide. Therefore, the CL method with high sensitivity could be established for detecting various substrates. A double-enzyme CL glucose biosensing platform was constructed for the determination of serum glucose employing the peroxidase-mimicking properties of Cu-MOF as well as glucose oxidase (GOx).


Assuntos
Técnicas Biossensoriais/métodos , Glicemia/análise , Cobre/química , Estruturas Metalorgânicas/química , Aspergillus niger/enzimologia , Catálise , Glucose Oxidase/química , Humanos , Peróxido de Hidrogênio/química , Limite de Detecção , Medições Luminescentes/métodos , Modelos Moleculares , Peroxidase/química
12.
Nat Commun ; 12(1): 3721, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140497

RESUMO

Cytosolic transport is an essential requirement but a major obstacle to efficient delivery of therapeutic peptides, proteins and nucleic acids. Current understanding of cytosolic delivery mechanisms remains limited due to a significant number of conflicting reports, which are compounded by low sensitivity and indirect assays. To resolve this, we develop a highly sensitive Split Luciferase Endosomal Escape Quantification (SLEEQ) assay to probe mechanisms of cytosolic delivery. We apply SLEEQ to evaluate the cytosolic delivery of a range of widely studied cell-penetrating peptides (CPPs) fused to a model protein. We demonstrate that positively charged CPPs enhance cytosolic delivery as a result of increased non-specific cell membrane association, rather than increased endosomal escape efficiency. These findings transform our current understanding of how CPPs increase cytosolic delivery. SLEEQ is a powerful tool that addresses fundamental questions in intracellular drug delivery and will significantly improve the way materials are engineered to increase therapeutic delivery to the cytosol.


Assuntos
Membrana Celular/metabolismo , Peptídeos Penetradores de Células/metabolismo , Citosol/metabolismo , Endossomos/metabolismo , Medições Luminescentes/métodos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Humanos , Luciferases/química , Espectrometria de Massas , Proteínas Recombinantes , Sensibilidade e Especificidade
13.
ACS Appl Mater Interfaces ; 13(25): 29392-29405, 2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34137577

RESUMO

Chemiluminescence immunoassays have been widely employed for diagnosing various diseases. However, because of the extremely low intensity chemiluminescence signals, highly sensitive transducers, such as photomultiplier tubes and image sensors with cooling devices, are required to overcome this drawback. In this study, a hypersensitive photosensor was developed based on cesium lead bromide (CsPbBr3) perovskite quantum dots (QDs) with sufficient high sensitivity for chemiluminescence immunoassays. First, CsPbBr3 QDs with a highly uniform size, that is, 5 nm, were synthesized under thermodynamic control to achieve a high size confinement effect. For the fabrication of the photosensor, MoS2 nanoflakes were used as an electron transfer layer and heat-treated at an optimum temperature. Additionally, a parylene-C film was used as a passivation layer to improve the physical stability and sensitivity of the photosensor. In particular, the trap states on the CsPbBr3 QDs were reduced by the passivation layer, and the sensitivity was increased. Finally, a photosensor based on CsPbBr3 QDs was employed in chemiluminescence immunoassays for the detection of human hepatitis B surface antigen, human immunodeficiency virus antibody, and alpha-fetoprotein (AFP, a cancer biomarker). When compared with the conventionally used equipment, the photosensor was determined to be feasible for application in chemiluminescence immunoassays.


Assuntos
Compostos de Cálcio/química , Imunoensaio/métodos , Chumbo/química , Medições Luminescentes/métodos , Óxidos/química , Pontos Quânticos/química , Titânio/química , Césio/química , Anticorpos Anti-HIV/análise , Antígenos de Superfície da Hepatite B/análise , Humanos , Polímeros/química , Xilenos/química
14.
Food Chem ; 362: 130219, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34091170

RESUMO

The use of artificial colorants in food is highly regulated due to their potential to harm human health. Thus, it is crucial to detect these substances effectively to ensure conformance with industrial standards. In this work, we prepared a photomultiplier tube (PMT)-based electrochemiluminescence (ECL) sensor and a charged coupled device (CCD)-based ECL sensor and compared their merits in the detection of sunset yellow (SY) dye. The sensors used C,N quantum dot-embedded g-C3N4 nanosheets (QDs@NSs) as the ECL agent and K2S2O8 as the coreactant. SY was analyzed on the basis of amplification in the QDs@NHs-K2S2O8 ECL system. The PMT-based sensor realized ultrasensitive detection using a single electrode, especially at low concentrations of SY. A CCD-based sensor imaged the ECL phenomenon of an electrode array and provided the advantages of high throughput and time savings. Under optimized conditions, both sensors exhibited high specificity, reproducibility and stability; detection limits of 20 nM with PMT detection and 5 µM with CCD detection were determined for SY, with detection ranging over at least two decades. The practical feasibilities of these systems were confirmed by satisfactory detection of SY in real drink samples.


Assuntos
Compostos Azo/análise , Bebidas Gaseificadas/análise , Técnicas Eletroquímicas/instrumentação , Corantes de Alimentos/análise , Compostos Azo/química , Técnicas Eletroquímicas/métodos , Eletrodos , Corantes de Alimentos/química , Limite de Detecção , Medições Luminescentes/instrumentação , Medições Luminescentes/métodos , Nanoestruturas , Pontos Quânticos , Reprodutibilidade dos Testes
15.
Methods Mol Biol ; 2274: 247-259, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050477

RESUMO

The present protocol demonstrates a novel mammalian cell imaging platform exerting a bioluminescence resonance energy transfer (BRET) system. This platform achieves a ~300 nm blue-to-near infrared shift of the emission (NIR-BRET) with the development of a unique coelenterazine (CTZ) derivative named BBlue2.3 and a fusion reporter protein probe named iRFP-RLuc8.6-535SG. The best NIR-BRET shift was achieved by tuning the blue emission peak of BBlue2.3 to a Soret band of the iRFP. In mammalian cells, BBlue2.3 emits light that is ~50-fold brighter than DeepBlueC in cell imaging when combined with RLuc8.6-535SG. This NIR-BRET platform is sufficiently brighter to be used for imaging live mammalian cells at single-cell level, and also for imaging metastases in deep tissues in live mice without generating considerable autoluminescence. This unique optical platform provides the brightest NIR-BLI template that can be used for imaging a diverse group of cellular events in living subjects.


Assuntos
Neoplasias da Mama/patologia , Transferência Ressonante de Energia de Fluorescência/métodos , Imidazóis/química , Luciferases/metabolismo , Medições Luminescentes/métodos , Imagem Óptica/métodos , Pirazinas/química , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Apoptose , Neoplasias da Mama/metabolismo , Proliferação de Células , Feminino , Humanos , Substâncias Luminescentes/química , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Methods Mol Biol ; 2274: 281-294, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050480

RESUMO

Optogenetic calcium sensors enable the imaging in real-time of the activities of single or multiple neurons in brain slices and in vivo. Bioluminescent probes engineered from the natural calcium sensor aequorin do not require illumination, are virtually devoid of background signal, and exhibit wide dynamic range and low cytotoxicity. These probes are thus well suited for long-duration, whole-field recordings of multiple neurons simultaneously. Here, we describe a protocol for monitoring and analyzing the dynamics of neuronal ensembles using whole-field bioluminescence imaging of an aequorin-based sensor in brain slice.


Assuntos
Equorina/química , Técnicas Biossensoriais/métodos , Encéfalo/metabolismo , Cálcio/metabolismo , Substâncias Luminescentes/química , Medições Luminescentes/métodos , Neurônios/metabolismo , Animais , Transferência Ressonante de Energia de Fluorescência/métodos , Camundongos , Vias Neurais , Imagem Óptica/métodos
17.
Methods Mol Biol ; 2255: 55-67, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033094

RESUMO

This chapter describes a real-time, bioluminescent apoptosis assay technique, which circumvents the well-documented "timing condundrum" encountered when employing traditional apoptosis detection chemistries after exposures with inducers of unknown potential. The assay continuously reports the translocation of phosphatidylserine (PS) from the inner membrane leaflet of a cell to the exofacial surface during apoptosis. This homogenous, no-wash, plate-based assay is made possible by two different annexin V fusion proteins, which contain complementing NanoBiT™ luciferase enzyme subunits, a time-released luciferase substrate, and a fluorescent membrane integrity reagent. During apoptosis, luminescence signal is proportional to PS exposure and fluorescence intensity correlated with the degree of secondary necrosis. Altogether, the measures provide exquisite kinetic resolution of dose- and agent-dependent apoptotic responses, from early through late phases. At exposure termination, other compatible reagents can be applied to measure additional orthogonal correlates of cell health.


Assuntos
Anexina A5/metabolismo , Apoptose , Neoplasias da Mama/patologia , Citometria de Fluxo/métodos , Leucemia Mieloide/patologia , Medições Luminescentes/métodos , Neoplasias da Mama/metabolismo , Feminino , Humanos , Leucemia Mieloide/metabolismo , Células Tumorais Cultivadas
18.
Methods Mol Biol ; 2255: 77-86, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033096

RESUMO

Three-dimensional (3D) in vitro systems closely resemble tissue microenvironments and provide predictive models for studying cytotoxic drug responses. The ability to capture the kinetic profiles of such responses in a dynamic and noninvasive way can further advance the utility of 3D cell cultures. Here, we describe the use of a luminescent lactate dehydrogenase (LDH) toxicity assay for monitoring time- and dose-dependent effects of drug treatment in 3D cancer spheroids. HCT116 spheroids formed in 96-well ultralow attachment plates were treated with increasing drug concentrations. Medium samples were collected at different timepoints, frozen, stored, and analyzed at the end of experiments using the luminescent LDH-Glo™ Assay. High assay sensitivity and low volume sampling enabled drug-induced toxicity profiling in a time- and dose-dependent manner.


Assuntos
Antineoplásicos/farmacologia , Digitonina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , L-Lactato Desidrogenase/metabolismo , Medições Luminescentes/métodos , Neoplasias/patologia , Esferoides Celulares/patologia , Testes de Toxicidade/métodos , Relação Dose-Resposta a Droga , Humanos , Indicadores e Reagentes/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Fatores de Tempo , Células Tumorais Cultivadas
19.
Methods Mol Biol ; 2255: 187-196, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033104

RESUMO

Comprehensive understanding of cellular responses to changes in the cellular environment or by drug treatment requires time-dependent analysis ranging from hours to several days. Here, we describe a sensitive, nonlytic live-cell assay that allows continuous or 'real-time' monitoring of cell viability, growth, and cytotoxicity over an extended period of time. We illustrate the use of the assay for small drug molecule and antibody-dependent cytotoxicity studies using cancer cells in 384-well plates. We show that the ability to measure changes in live cells over time provides instantaneous information on the biological status of the cells, information about the mode of action of the drug, and offers an added advantage of preserving the cells for multiplexing with downstream applications.


Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Apoptose , Bioensaio/métodos , Neoplasias da Mama/patologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Medições Luminescentes/métodos , Neoplasias da Mama/metabolismo , Feminino , Humanos , Células Tumorais Cultivadas
20.
Methods Mol Biol ; 2274: 37-42, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050460

RESUMO

The current standard murine model of bone metastasis by using intracardiac injection (IC) has some limitations despite the great utility of this model. This fact emphasizes the need for a new murine model to accelerate basic research of bone metastasis. The present protocol provides instructions on caudal artery (CA) injection that is an easy-to-use method to reliably construct a murine bone metastasis model with a variety type of cancer cell lines. Bioluminescence imaging visualized that cancer cells injected via the caudal artery in the tail were efficiently delivered to a hind limb bone, where it is a common site affected with bone metastasis in mice. CA injection rarely causes stress-induced acute death in mice and enables us to inject a large number of cancer cells, thereby greatly increasing the frequency of bone metastasis in hind limb bones. Importantly, CA injection is technically as easy as tail vein injection and causes no lethal stress, indicating that it is a model that also contributes to animal welfare. CA injection model, therefore, could represent a powerful tool for many researchers to study molecular mechanisms of bone metastasis in mice.


Assuntos
Neoplasias Ósseas/secundário , Carcinoma Pulmonar de Lewis/patologia , Artérias Carótidas/patologia , Processamento de Imagem Assistida por Computador/métodos , Medições Luminescentes/métodos , Animais , Neoplasias Ósseas/diagnóstico por imagem , Carcinoma Pulmonar de Lewis/diagnóstico por imagem , Artérias Carótidas/diagnóstico por imagem , Camundongos
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