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1.
Anal Bioanal Chem ; 411(19): 4951-4961, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30982928

RESUMO

Immunomagnetic separation (IMS) was combined with flow-based chemiluminescence sandwich immunoassays (CL-SIA) for the quantification of Staphylococcal enterotoxin B in milk. Therefore, iron oxide-shell silica-core magnetic nanocomposites were conjugated to biotinylated anti-SEB antibodies (MNC-IgGs). MNC-IgGs were applied successfully for (i) capturing SEB in milk samples by an affinity reaction, (ii) magnetophoretic collection on antibody spots in a channel of a flow-based immunochip, and (iii) sensitive enzymatic chemiluminescence detection of biotin labels by poly(horseradish peroxidase)-streptavidin. IMS was performed in 0.6 mL and 100 mL milk samples resulting in detection limits of 50 ng L-1 and 0.39 ng L-1, respectively, for the combined analytical method. It was shown that the assay sensitivity was dramatically improved by the combination of IMS with flow-based CL-SIA compared to CL-SIA directly applied with milk samples (detection limit 130 ng L-1). The IMS-CL-SIA has a time-to-result of 2-3 h. The reported combined analytical method can be used for a rapid control of SEB in complex food matrices such as milk. In future, even the monitoring of multiple contaminants in food or water may be performed by IMS-CL-SIA. Graphical abstract.


Assuntos
Enterotoxinas/análise , Imunoensaio/métodos , Separação Imunomagnética/instrumentação , Luminescência , Magnetismo , Leite/química , Nanocompostos/química , Staphylococcus aureus/química , Superantígenos/análise , Animais , Automação , Biotina/análise , Microbiologia de Alimentos/métodos , Peroxidase do Rábano Silvestre/química , Limite de Detecção , Estreptavidina/química
2.
Biosens Bioelectron ; 129: 175-181, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30710755

RESUMO

White blood cells (WBCs) isolated from peripheral blood have been verified as important biomarkers for the diagnosis, treatment and prognosis of cancer. However, it's still under challenge to acquire high-purity WBCs, even by taking advantage of current microfluidic technology. Considering the universality of clinical magnetic activated cell sorting (MACS) method, new developments on microfluidic chip in combination of magnetic cells separation technologies may provide a fascinating approach for high-purity WBCs sorting and widely clinical application. Here, we present a flyover style microfluidic chip which has been elaborately embedded with two-stage magnetic separation in continuous flow for WBCs sorting. Immunomagnetic micro/nano-particles (IMNPs) labeled WBC (WBC@IMNPs) were sequentially separated by a lateral magnetic force and a vertical magnetic force, and the final separation purity of WBCs reached up to 93 ±â€¯1.67% at a flow rate of 20 µL min-1. Furthermore, the WBCs viability was up to 97.5 ±â€¯1.8%. Consequently, this novel flyover style microfluidic-chip with magnetic separation technology has been successfully demonstrated as cut-in-edge method for high-purity WBCs sorting, and obviously it's easy to extend for other types of cells sorting under great potential application in biomedical fields.


Assuntos
Separação Imunomagnética/instrumentação , Dispositivos Lab-On-A-Chip , Leucócitos/citologia , Animais , Sobrevivência Celular , Desenho de Equipamento , Campos Magnéticos , Camundongos Endogâmicos BALB C , Níquel/química
3.
Methods Mol Biol ; 1884: 151-160, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30465201

RESUMO

Characterization of individual cell populations from the tumor microenvironment is critical to understand their functional contribution to tumor progression. Magnetic bead enrichment and fluorescence-activated cell sorting (FACS) allow for the isolation of specific cell types that can be used in downstream applications, including in vitro and in vivo functional studies and molecular profiling. In this chapter, we describe the process of isolation of tumor-associated macrophages (TAMs) from primary murine breast tumors subsequent to therapeutic or experimental intervention. Additionally, we further detail how to analyze their ability to support tumor cell growth by co-injecting isolated TAMs with tumor cells orthotopically into the mammary gland of immune-deficient hosts, and monitoring tumor progression by live imaging and caliper measurement.


Assuntos
Separação Celular/métodos , Citometria de Fluxo/métodos , Separação Imunomagnética/métodos , Macrófagos/imunologia , Neoplasias Mamárias Experimentais/imunologia , Animais , Antígeno CD11b/imunologia , Antígeno CD11b/metabolismo , Separação Celular/instrumentação , Transformação Celular Neoplásica/imunologia , Feminino , Citometria de Fluxo/instrumentação , Corantes Fluorescentes/química , Separação Imunomagnética/instrumentação , Macrófagos/metabolismo , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Microambiente Tumoral/imunologia
4.
Methods Mol Biol ; 1884: 231-245, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30465207

RESUMO

Exosomes are membrane-enclosed vesicles released by different cell types into the extracellular space. As mediators of intercellular communication, they are involved in multiple physiological processes, but they are also associated with the pathogenesis of human malignancies including leukemia. Isolation of exosomes enables the characterization of their role in microenvironment modulation as well as their participation in disease pathology. A variety of strategies and techniques exists to purify exosomes from many biological fluids (e.g., blood, urine, and saliva). Here, we describe the efficient production of large quantities of exosomes from leukemic cell lines by using CELLine bioreactors based on two-compartment technology, as well as their isolation and purification by combining differential centrifugation and ultracentrifugation through a density gradient (17% OptiPrep™ cushion). Thus, exosomes are appropriately prepared for characterization by western blotting to detect exosome markers or imaging flow cytometry (ImageStream), and for downstream analyses such as the internalization in microenvironmental cells by confocal imaging or flow cytometry, methods which are also described in this chapter.


Assuntos
Reatores Biológicos , Técnicas de Cultura de Células/instrumentação , Fracionamento Celular/métodos , Exossomos/patologia , Leucemia/patologia , Técnicas de Cultura de Células/métodos , Fracionamento Celular/instrumentação , Linhagem Celular Tumoral , Centrifugação com Gradiente de Concentração/instrumentação , Centrifugação com Gradiente de Concentração/métodos , Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Corantes Fluorescentes/química , Humanos , Separação Imunomagnética/instrumentação , Separação Imunomagnética/métodos , Leucemia/imunologia , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Microambiente Tumoral/imunologia
5.
Methods Mol Biol ; 1884: 247-258, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30465208

RESUMO

Cancer cells leaving the primary tumor immunosuppressive microenvironment become vulnerable to active immune surveillance and require mechanisms of immunoevasion to survive in the circulation. Studies have identified several pathways by which circulating tumor cells (CTCs) might escape the immune system/immunotherapy attack. The PD-1/PD-L1 axis is an immune checkpoint regulator, playing a major role in maintaining self-tolerance. It is now well recognized that tumor cells co-opt the PD-1/PD-L1 axis of immune regulation to interfere with cytotoxic T lymphocyte function. Transcriptional changes in CTCs, leading to the upregulation of PD-L1, might enable them to survive in circulation. Very recent data revealed a previously unappreciated role of epithelial-mesenchymal transition (EMT) in reprogramming the immune response in the local tumor microenvironment and a mutual regulation between EMT and immunoevasion is becoming apparent. In this chapter, we will describe in detail both EpCAM-dependent and -independent approaches that allow the identification of PD-L1 expression and EMT-like features in circulating tumor cells.


Assuntos
Separação Imunomagnética/métodos , Neoplasias/imunologia , Células Neoplásicas Circulantes/patologia , Evasão Tumoral/imunologia , Antígeno B7-H1/imunologia , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Molécula de Adesão da Célula Epitelial/imunologia , Molécula de Adesão da Célula Epitelial/metabolismo , Transição Epitelial-Mesenquimal/imunologia , Humanos , Separação Imunomagnética/instrumentação , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Neoplasias/sangue , Neoplasias/patologia , Células Neoplásicas Circulantes/imunologia , Receptor de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T/imunologia , Linfócitos T/patologia , Microambiente Tumoral/imunologia
6.
Biomed Microdevices ; 20(4): 99, 2018 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-30417219

RESUMO

We demonstrate enhanced on-chip circulating tumor cell (CTC) detection through the incorporation of plasmonic-enhanced near-infrared (NIR) fluorescence screening. Specifically, the performance of plasmonic gold coated chips was evaluated on our previously reported immunomagnetic CTC capture system and compared to the performance of a regular chip. Three main performance metrics were evaluated: capture efficiency, capture reproducibility, and clinical efficacy. Use of the plasmonic chip to capture SK-BR-3 cells in PBS, resulted in a capture efficiency of 82%, compared to 76% with a regular chip. Both chips showed excellent capture reproducibility for all three cells lines evaluated (MCF-7, SK-BR-3, Colo 205) in both PBS and peripheral blood, with R2 values ranging from 0.983 to 0.996. Finally, performance of the plasmonic chip was evaluated on thirteen peripheral blood samples in patients with both breast and prostate cancer. The regular chip detected 2-8 cells per 5 mL of blood, while the plasmonic chip detected 8-85 cells per 5 mL of blood in parallel samples. In summary, we successfully demonstrate improved CTC capture and detection capabilities through use of plasmonic-enhanced near-infrared (NIR) fluorescence screening in both in vitro and ex vivo experiments. This work not only has the potential to improve clinical outcomes though improved CTC analysis, but also demonstrates successful interface design between plasmonic materials and cell capture for bioanalytical applications.


Assuntos
Separação Imunomagnética/instrumentação , Dispositivos Lab-On-A-Chip , Limite de Detecção , Células Neoplásicas Circulantes/patologia , Adulto , Idoso , Feminino , Humanos , Células MCF-7 , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes
7.
Lab Chip ; 18(24): 3830-3839, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30394473

RESUMO

Rapid, sensitive analysis of protein biomarkers is of tremendous biological and clinical significance. Immunoassays are workhorse tools for protein analysis and have been under continuous investigation to develop new methods and to improve the analytical performance. Herein we report a pneumatically gated microfluidic communicating vessel (µCOVE) chip for rapid and sensitive immunomagnetic ELISA. A distinct feature of our device is that it employs the communicating vessel principle as a simple means to generate a fast transient hydrodynamic flow to enable effective flow washing without the need for excessive incubation, which greatly simplifies and expedites the assay workflow, compared to conventional microfluidic flow-based immunoassays. Stationary multi-phase microfluidic techniques have been developed for fast bead washing. However, they have some limitations, such as the need for careful control of interfacial properties, large bead quantity required for reliable interphase bead transport, and relatively high bead loss during surface tension-gated traverse. Our single-phase µCOVE chip can overcome such limitations and facilitate the manipulation of magnetic beads to streamline the assay workflow. We showed that the µCOVE device affords highly sensitive quantification of the CEA and EGFR proteins with a LOD down to the sub-picogram per mL level. Direct detection of the EGFR in the crude A431 cell lysate was also demonstrated to further validate the ability of our device for rapid and quantitative analysis of complex biological samples. Overall, our work presents a unique platform that combines the merits of the stationary multi-phase systems and the flow-based microfluidics. This novel immunoassay microsystem has promising potential for a broad range of biological and clinical applications, owing to its simplicity and high performance.


Assuntos
Separação Imunomagnética/instrumentação , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Anticorpos/metabolismo , Linhagem Celular Tumoral , Desenho de Equipamento , Receptores ErbB/análise , Receptores ErbB/metabolismo , Humanos , Separação Imunomagnética/métodos
8.
Angew Chem Int Ed Engl ; 57(45): 14942-14946, 2018 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-30187617

RESUMO

A combination of an immuno-affinity enrichment strategy and sensitive amperometric read-out was implemented in a point-of-care platform intended for bacterial infection analysis. Bacterial cells, selectively captured and enriched from complex matrices through immuno-affinity, were detected by amperometric monitoring of the redox state of metabolic activity indicators, providing species identification and viable-cell quantification. The method was successfully employed for the diagnosis of bacterial infections including antimicrobial susceptibility testing with only several hours of total working time.


Assuntos
Anticorpos Imobilizados/química , Bactérias/isolamento & purificação , Infecções Bacterianas/diagnóstico , Técnicas Biossensoriais/instrumentação , Técnicas Eletroquímicas/instrumentação , Separação Imunomagnética/instrumentação , Sistemas Automatizados de Assistência Junto ao Leito , Desenho de Equipamento , Humanos , Dispositivos Lab-On-A-Chip
9.
Appl Microbiol Biotechnol ; 102(20): 8931-8942, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30143837

RESUMO

Single-domain antibody (sdAb) or nanobody possesses specific features non-accessible for conventional antibodies that make them suitable for research and biotechnological applications. Porcine epidemic diarrhea virus (PEDV) causes lethal diarrhea in piglets, resulting in great economic losses all over the world. To detect and isolate PEDV rapidly and accurately is important for the control and further research of the clinical PEDV strains. In this study, four sdAb fragments (sdAb-Mc19/29/30/37) targeting the membrane (M) protein of PEDV were selected from sdAb library that was constructed through M protein-immunized Camelus bactrianus. The selected sdAb-Mcs were solubly expressed in Escherichia coli. The functional characteristics analysis revealed that the recombinant sdAb-Mcs have excellent binding activity and specificity to M protein but have no neutralizing activity to PEDV. For further application, sdAb-Mc37 was conjugated with quantum dots to synthesize a nanoprobe for imaging PEDV in vero cells. The observed fluorescence in vero cells clearly reflects that PEDV virions can be reliably recognized and labeled by the nanoprobe. Furthermore, the sdAb-Mc29 was conjugated with superparamagnetic nanobeads to construct immunomagnetic nanobeads (IMNBs) used to isolate PEDV. One PEDV strain was successfully isolated from clinical fecal sample, suggesting IMNBs as a novel and efficient tool suitable for PEDV isolation from clinical samples. This study provided a novel application and substantiated the suitability of sdAb as a specific binder for the isolation of viruses.


Assuntos
Anticorpos Antivirais/química , Infecções por Coronavirus/veterinária , Diarreia/veterinária , Separação Imunomagnética/métodos , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Anticorpos de Domínio Único/química , Doenças dos Suínos/virologia , Animais , Anticorpos Antivirais/genética , Anticorpos Antivirais/metabolismo , Camelus , Infecções por Coronavirus/virologia , Diarreia/virologia , Fluorescência , Imunização , Separação Imunomagnética/instrumentação , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/imunologia , Pontos Quânticos/química , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/metabolismo , Suínos , Células Vero
10.
J Immunoassay Immunochem ; 39(3): 308-322, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29995570

RESUMO

Fe3O4/Ag core/shell nanoparticles functionalized with the free amino (NH2) functional groups (Fe3O4/Ag-NH2) were conjugated with fluorescent electron coupled dye (ECD)-antiCD34 antibody using the 1-ethyl-3-(3'-dimethyl-aminopropyl) carbodiimide (EDC) catalyst (ECD - Electron Coupled Dye or R Phycoerythrin-Texas Red is a fluorescent organic dye attached to the antibody). The characteristic fluorescence of ECD in the antibody was investigated and was used as a good indicator for estimating the percentage of the antibodies that were successfully conjugated with the nanoparticles. The conjugation efficiency was found to increase depending on the VNP:VAB ratio, where VNP and VAB are the volumes of the nanoparticle solution (concentration of 50 ppm) and the as-purchased antibody solution, respectively. The conjugation efficiency rapidly increased from approximately 18% to approximately 70% when VNP:VAB was increased from 2:1 to 100:1, and it gradually reached the saturated state at an efficiency of 95%, as the VNP:VAB was equal to 300:1. The bioactivity of the abovementioned conjugation product (denoted by Fe3O4/Ag-antiCD34) was evaluated in an experiment for the collection of stem cells from bone marrow samples.


Assuntos
Antígenos CD34/análise , Separação Celular/métodos , Óxido Ferroso-Férrico/química , Separação Imunomagnética/métodos , Nanopartículas/química , Prata/química , Células-Tronco/citologia , Antígenos CD34/imunologia , Separação Celular/instrumentação , Humanos , Separação Imunomagnética/instrumentação , Células-Tronco/imunologia
11.
Lab Chip ; 18(14): 1997-2002, 2018 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-29923569

RESUMO

Microfluidics brings unique opportunities for the synthesis of nanomaterials toward efficient liquid biopsy. Herein, we developed the microreactor-enabled flow synthesis of immunomagnetic nanomaterials with controllable shapes (sphere, cube, rod, and belt) by simply tuning the flow rates. The particle shape-dependent screening efficiency of circulating tumor cells was first investigated and compared with commercial ferrofluids, providing new insights into the rational design of a particulate system toward the screening and analysis of circulating tumor biomarkers.


Assuntos
Separação Imunomagnética/instrumentação , Dispositivos Lab-On-A-Chip , Biópsia Líquida/instrumentação , Nanoestruturas , Humanos , Células MCF-7 , Células Neoplásicas Circulantes/patologia
12.
J Agric Food Chem ; 66(19): 4941-4947, 2018 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-29709176

RESUMO

Here, we report an enhanced colorimetric method using enzymatic amplification with nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) precipitation for the ultrasensitive detection of Escherichia coli O157:H7 through immunomagnetic separation-selective filtration. Biotinylated anti- E. coli O157:H7 antibody and streptavidin-alkaline phosphatase were conjugated to the surface of magnetic nanoparticles, and E. coli O157:H7-conjugates complexes remained on the membrane filter surface. The resultant light brown spots on the membrane filter were amplified with NBT/BCIP solution to yield enzyme-catalyzed precipitation, which increased with an increasing E. coli O157:H7 concentration. E. coli O157:H7 was detected in pure samples with limits of detection of 10 and 6.998 colony-forming units (CFU)/mL through visual observation and measurement of optical density, respectively. The proposed method was applied to a lettuce sample inoculated with selective E. coli O157:H7, which was detected within 55 min without cross-reactivity to non-target bacteria. This enhanced colorimetric method has potential for on-site detection of food contaminants and environmental pollutants.


Assuntos
Escherichia coli O157/isolamento & purificação , Filtração/métodos , Separação Imunomagnética/métodos , Contagem de Colônia Microbiana , Colorimetria , Escherichia coli O157/crescimento & desenvolvimento , Contaminação de Alimentos/análise , Separação Imunomagnética/instrumentação , Indóis/química , Alface/microbiologia , Nitroazul de Tetrazólio/química , Sensibilidade e Especificidade
13.
J Immunol Methods ; 458: 74-82, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29684430

RESUMO

Cell-based assays to monitor antigen-specific T-cell responses are characterized by their high complexity and should be conducted under controlled conditions to lower multiple possible sources of assay variation. However, the lack of standard reagents makes it difficult to directly compare results generated in one lab over time and across institutions. Therefore TCR-engineered reference samples (TERS) that contain a defined number of antigen-specific T cells and continuously deliver stable results are urgently needed. We successfully established a simple and robust TERS technology that constitutes a useful tool to overcome this issue for commonly used T-cell immuno-assays. To enable users to generate large-scale TERS, on-site using the most commonly used electroporation (EP) devices, an RNA-based kit approach, providing stable TCR mRNA and an optimized manufacturing protocol were established. In preparation for the release of this immuno-control kit, we established optimal EP conditions on six devices and initiated an extended RNA stability study. Furthermore, we coordinated on-site production of TERS with 4 participants. Finally, a proficiency panel was organized to test the unsupervised production of TERS at different laboratories using the kit approach. The results obtained show the feasibility and robustness of the kit approach for versatile in-house production of cellular control samples.


Assuntos
Bioensaio/normas , Engenharia Celular/métodos , RNA Mensageiro/metabolismo , Receptores de Antígenos Quiméricos/genética , Linfócitos T/imunologia , Bioensaio/métodos , Buffy Coat/citologia , Técnicas de Cultura de Células/métodos , Engenharia Celular/instrumentação , Eletroporação/instrumentação , Eletroporação/métodos , Estudos de Viabilidade , Antígeno HLA-A2/imunologia , Humanos , Separação Imunomagnética/instrumentação , Separação Imunomagnética/métodos , Estabilidade de RNA , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Padrões de Referência , Linfócitos T/metabolismo
14.
Cytotherapy ; 20(4): 532-542, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29500069

RESUMO

BACKGROUND AIMS: For patients needing allogeneic stem cell transplantation but lacking a major histocompatibility complex (MHC)-matched donor, haplo-identical (family) donors may be an alternative. Stringent T-cell depletion required in these cases to avoid lethal graft-versus-host disease (GVHD) can delay immune reconstitution, thus impairing defense against virus reactivation and attenuating graft-versus-leukemia (GVL) activity. Several groups reported that GVHD is caused by cells residing within the naive (CD45RA+) T-cell compartment and proposed use of CD45RA-depleted donor lymphocyte infusion (DLI) to accelerate immune reconstitution. We developed and tested the performance of a CD45RA depletion module for the automatic cell-processing device CliniMACS Prodigy and investigated quality attributes of the generated products. METHODS: Unstimulated apheresis products from random volunteer donors were depleted of CD45RA+ cells on CliniMACS Prodigy, using Good Manufacturing Practice (GMP)-compliant reagents and methods throughout. Using phenotypic and functional in vitro assays, we assessed the cellular constitution of CD45RA-depleted products, including T-cell subset analyses, immunological memory function and allo-reactivity. RESULTS: Selections were technically uneventful and proceeded automatically with minimal hands-on time beyond tubing set installation. Products were near-qualitatively CD45RA+ depleted, that is, largely devoid of CD45RA+ T cells but also of almost all B and natural killer cells. Naive and effector as well as γ/δ T cells were greatly reduced. The CD4:CD8 ratio was fivefold increased. Mixed lymphocyte reaction assays of the product against third-party leukocytes revealed reduced allo-reactivity compared to starting material. Anti-pathogen responses were retained. DISCUSSION: The novel, closed, fully GMP-compatible process on Prodigy generates highly CD45RA-depleted cellular products predicted to be clinically meaningfully depleted of GvH reactivity.


Assuntos
Doença Enxerto-Hospedeiro/prevenção & controle , Memória Imunológica/fisiologia , Imunoterapia Adotiva , Antígenos Comuns de Leucócito/metabolismo , Depleção Linfocítica , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/transplante , Adulto , Automação Laboratorial , Células Cultivadas , Feminino , Doença Enxerto-Hospedeiro/imunologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/métodos , Teste de Histocompatibilidade , Humanos , Separação Imunomagnética/instrumentação , Separação Imunomagnética/métodos , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Leucaférese/instrumentação , Leucaférese/métodos , Teste de Cultura Mista de Linfócitos , Depleção Linfocítica/instrumentação , Depleção Linfocítica/métodos , Masculino , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Doadores de Tecidos , Transplante Homólogo , Adulto Jovem
15.
Methods Mol Biol ; 1756: 177-186, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29600370

RESUMO

While circulating tumor cells (CTCs) within peripheral blood of cancer patients are no new phenomenon in many carcinomas, there is a lack of information on the biological and clinical implications of CTCs in esophageal adenocarcinomas. Limited evidence suggests that the CTCs are frequently detected in esophageal adenocarcinomas when compared to esophageal squamous cell carcinoma suggesting the potential difference in the pathogenesis between these two carcinomas. In addition, the varied CTC levels between adenocarcinoma and squamous cell carcinomas of the esophagus could be attributed to the varied expression pattern of epithelial markers such as epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK). In esophageal adenocarcinomas, CTC levels correlated with pathological T stages, lymph node metastasis, and patient survival. Thus, detection of CTCs potentially acts as a noninvasive and real-time biomarker for predicting patient prognosis in esophageal adenocarcinomas. Although the CTC detection is currently performed using various methods, the only Food and Drug Administration (FDA) of USA approved CTC detection method in clinics is the CELLSEARCH® system. This chapter will discuss various biological characteristics of CTC and its potential implications in esophageal adenocarcinomas. In addition, a quick overview of CTC detection methodology is outlined.


Assuntos
Adenocarcinoma/sangue , Biomarcadores Tumorais/análise , Neoplasias Esofágicas/sangue , Separação Imunomagnética/métodos , Células Neoplásicas Circulantes/patologia , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adenocarcinoma/terapia , Biomarcadores Tumorais/imunologia , Contagem de Células/instrumentação , Contagem de Células/métodos , Linhagem Celular Tumoral , Molécula de Adesão da Célula Epitelial/imunologia , Neoplasias Esofágicas/mortalidade , Neoplasias Esofágicas/patologia , Neoplasias Esofágicas/terapia , Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Humanos , Separação Imunomagnética/instrumentação , Estadiamento de Neoplasias/métodos , Células Neoplásicas Circulantes/imunologia , Células Neoplásicas Circulantes/metabolismo , Prognóstico , Taxa de Sobrevida , Resultado do Tratamento
16.
Adv Exp Med Biol ; 1029: 101-107, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29542083

RESUMO

Genome-wide studies in Ciona often require highly purified cell populations. In this methods chapter, we introduce multi-channel combinatorial fluorescence activated cells sorting (FACS) and magnetic-activated cell sorting (MACS) as two sensitive and efficient tools for isolating lineage-specific cell populations from dissociated Ciona embryos and larvae. We present isolation of trunk ventral cell (TVC) progeny as the test case most commonly used in our laboratory. These approaches may also be applied to purify other cell populations with the proper combination of tissue-specific reporters.


Assuntos
Ciona intestinalis/embriologia , Citometria de Fluxo/métodos , Genes Reporter , Separação Imunomagnética/métodos , Proteínas Luminescentes/análise , Animais , Linhagem da Célula , Ciona intestinalis/citologia , Ciona intestinalis/genética , Técnicas de Cultura Embrionária , Embrião não Mamífero/química , Embrião não Mamífero/citologia , Elementos Facilitadores Genéticos , Citometria de Fluxo/instrumentação , Separação Imunomagnética/instrumentação , Mosaicismo , RNA/isolamento & purificação
17.
Methods Mol Biol ; 1753: 305-315, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29564798

RESUMO

microRNA profiling has identified cell-specific expression patterns that could represent molecular signatures triggering the acquisition of a specific phenotype; in other words, of cellular identity and its associated function. Several groups have hypothesized that retinal cell phenotyping could be achieved through the determination of the global pattern of miRNA expression across specific cell types in the adult retina. This is especially relevant for Müller glia in the context of retinal damage, as these cells undergo dramatic changes of gene expression in response to injury, that render them susceptible to acquire a progenitor-like phenotype and be a source of new neurons.We describe a method that combines an experimental protocol for excitotoxic-induced retinal damage through N-methyl-D-aspartate subretinal injection with magnetic-activated cell sorting (MACS) of Müller cells and RNA isolation for microRNA profiling. Comparison of microRNA patterns of expression should allow Müller cell phenotyping under different experimental conditions.


Assuntos
Células Ependimogliais/metabolismo , Perfilação da Expressão Gênica/métodos , Separação Imunomagnética/métodos , MicroRNAs/metabolismo , Doenças Retinianas/patologia , Animais , Modelos Animais de Doenças , Células Ependimogliais/efeitos dos fármacos , Células Ependimogliais/patologia , Transportador 1 de Aminoácido Excitatório/imunologia , Perfilação da Expressão Gênica/instrumentação , Humanos , Separação Imunomagnética/instrumentação , Injeções Intravítreas , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/isolamento & purificação , N-Metilaspartato/administração & dosagem , N-Metilaspartato/toxicidade , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Doenças Retinianas/induzido quimicamente , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação
18.
Food Microbiol ; 72: 23-30, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29407402

RESUMO

Cronobacter species are foodborne pathogens that can affect the human central nervous system. Survivors of Cronobacter infections often suffer from severe neurological impairments, including hydrocephalus, quadriplegia, and developmental delays in all ages, especially in infants and the immunocompromised. Moreover, Cronobacter species pose a high risk in powdered infant formula (PIF) because PIF is a major source of nutrition for infants worldwide. To develop a rapid and sensitive detection method for Cronobacter species in PIF, immunoliposomes and immunomagnetic nanoparticles were synthesized, after which an immunoliposome-based immunomagnetic concentration and separation assay was developed and applied to PIF for the detection of Cronobacter species. The detection limits of the developed assay were 5.9 × 103 ± 0.7-4.8 × 104 ± 0.2 CFU/mL for Cronobacter species in pure culture with no cross-reactivity with 13 other tested non-Cronobacter strains. Additionally, the developed assay could provide results in 3 h when the contaminated level was higher than 104 CFU/25 g PIF and in 9 h when the contaminated level was 10 CFU/25 g PIF. The developed immunoliposome-based immunomagnetic concentration and separation assay is rapid, sensitive, and simple and thus has great potential for use in the detection of Cronobacter species in PIF.


Assuntos
Cronobacter/isolamento & purificação , Contaminação de Alimentos/análise , Separação Imunomagnética/métodos , Fórmulas Infantis/microbiologia , Cronobacter/classificação , Cronobacter/genética , Humanos , Separação Imunomagnética/instrumentação , Fórmulas Infantis/análise , Pós/análise
19.
Int J Food Microbiol ; 267: 1-8, 2018 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-29275279

RESUMO

Immunomagnetic separation (IMS) as a culture-free enrichment sample preparation technique has gained increasing popularity in the development of rapid detection methods for foodborne pathogens. While the use of magnetic nanoparticles in IMS is on the rise due to substantially larger surface area compared to conventional magnetic microparticles, the effects of immunomagnetic bead (IMB) size on pathogen cell recovery are not fully understood. In this study we used IMBs of different sizes (100, 500, and 1000nm diameters) to capture Salmonella Enteritidis, a common foodborne pathogen, from buffer solutions as well as food matrices (chicken carcass rinse and liquid egg white). The IMS recovery and non-specific binding rate were compared. The recoveries of Salmonella cells in buffers was highest using the 100nm IMBs (88-96%), followed by the 500nm (31-89%) and 1000nm (4.1-61%) IMBs, demonstrating a significant size effect. The non-specific binding rates of E. coli also increased as IMB size decreased. A 2-72% reduction in Salmonella recovery was observed in chicken carcass rinse and liquid egg white samples compared to in buffers, and this reduction was more significant using 500 and 1000nm IMBs. However, lower IMS recoveries (10-56%) was found in 100nm IMBs two months after preparation. Overall, magnetic nanoparticles yielded superior IMS efficiency to micrometer size IMBs and were less subjective to interference from food matrices. Nevertheless, their long term stability remains an obstacle towards successful use in IMS.


Assuntos
Escherichia coli/isolamento & purificação , Microbiologia de Alimentos/métodos , Separação Imunomagnética/instrumentação , Separação Imunomagnética/métodos , Tamanho da Partícula , Salmonella enteritidis/isolamento & purificação , Animais , Galinhas/microbiologia , Clara de Ovo/microbiologia , Microbiologia de Alimentos/normas
20.
Electrophoresis ; 39(3): 526-533, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28868639

RESUMO

The success of microfluidic immunocapture based on magnetic beads depends primarily on a sophisticated microscale separation system and on the quality of the magnetic immunosorbent. A microfluidic chip containing a magnetically stabilized fluidized bed (µMSFB), developed for the capture and on-chip amplification of bacteria, was recently described by Pereiro et al.. The present work shows the thorough development of anti-Salmonella magnetic immunosorbents with the optimal capture efficiency and selectivity. Based on the corresponding ISO standards, these parameters have to be high enough to capture even a few cells of bacteria in a proper aliquot of sample, e.g. milk. The selection of specific anti-Salmonella IgG molecules and the conditions for covalent bonding were the key steps in preparing an immunosorbent of the desired quality. The protocol for immunocapturing was first thoroughly optimized and studied in a batchwise arrangement, and then the carrier was integrated into the µMSFB chip. The combination of the unique design of the chip (guaranteeing the collision of cells with magnetic beads) with the advanced immunosorbent led to a Salmonella cell capture efficiency of up to 99%. These high values were achieved repeatedly even in samples of milk differing in fat content. The rate of nonspecific capture of Escherichia coli (i.e. the negative control) was only 2%.


Assuntos
Separação Imunomagnética/métodos , Leite/química , Salmonella/isolamento & purificação , Animais , Escherichia coli/isolamento & purificação , Imunoglobulina G/química , Separação Imunomagnética/instrumentação , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Microesferas , Salmonella/citologia , Salmonella/imunologia
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