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1.
Braz. J. Biol. ; 83: 1-8, 2023. tab, graf
Artigo em Inglês | VETINDEX, VETINDEX | ID: vti-765533

RESUMO

Hybridization and Polyploidization are most common of the phenomenon observed in plants, especially in the genus Nicotiana leading to the duplication of genome. Although genomic changes associated with these events has been studied at various levels but the genome size and GC content variation is less understood because of absence of sufficient genomic data. In this study the flow cytometry technique was used to uncover the genome size and GC contents of 46 Nicotiana species and we compared the genomic changes associated with the hybridization events along evolutionary time scale. The genome size among Nicotiana species varied between 3.28 pg and 11.88 pg whereas GC contents varied between 37.22% and 51.25%. The tetraploid species in genus Nicotiana including section Polydiclae, Repandae, Nicotiana, Rustica and Sauveolentes revealed both up and downsizing in their genome sizes when compared to the sum of genomes of their ancestral species. The genome sizes of three homoploid hybrids were found near their ancestral species. Loss of large genome sequence was observed in the evolutionary more aged species (>10 Myr) as compared to the recently evolved ones (<0.2 Myr). The GC contents were found homogenous with a mean difference of 2.46% among the Nicotiana species. It is concluded that genome size change appeared in either direction whereas the GC contents were found more homogenous in genus Nicotiana.(AU)


A hibridização e a poliploidização são os fenômenos mais comuns observados em plantas, principalmente no gênero Nicotiana, levando à duplicação do genoma. Embora as mudanças genômicas associadas a esses eventos tenham sido estudadas em vários níveis, o tamanho do genoma e a variação do conteúdo de GC são menos compreendidos devido à ausência de dados genômicos suficientes. Neste estudo, a técnica de citometria de fluxo foi usada para descobrir o tamanho do genoma e o conteúdo de GC de 46 espécies de Nicotiana, e comparamos as mudanças genômicas associadas aos eventos de hibridização ao longo da escala de tempo evolutiva. O tamanho do genoma entre as espécies de Nicotiana variou entre 3,28 pg e 11,88 pg, enquanto os conteúdos de GC variaramentre 37,22% e 51,25%. As espécies tetraploides do gênero Nicotiana, incluindo as seções Polydiclae, Repandae, Nicotiana, Rustica e Sauveolentes, revelaram aumento e redução do tamanho do genoma quando comparados à soma dos genomas de suas espécies ancestrais. Os tamanhos do genoma de três híbridos homoploides foram encontrados perto de suas espécies ancestrais. A perda da grande sequência do genoma foi observada nas espécies evolutivas mais velhas (> 10 Myr) em comparação com as que evoluíram recentemente (< 0,2 Myr). Os teores de GC foram homogêneos com diferença média de 2,46% entre as espécies de Nicotiana. Conclui-se que a mudança no tamanho do genoma apareceu em ambas as direções, enquanto os conteúdos de GC foram encontrados mais homogêneos no gênero Nicotiana.(AU)


Assuntos
Tabaco/genética , Genoma , Tamanho do Genoma , Citometria de Fluxo/métodos , Separação Celular/métodos
2.
STAR Protoc ; 3(2): 101337, 2022 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-35496783

RESUMO

Understanding macrophage heterogeneity in tissue repair is a major challenge. Here, we describe a protocol that combines isolation of immune cells from skin wounds with subsequent flow-cytometry-based sorting of wound macrophages and single-cell RNA sequencing. We use a modified version of the original Smart-seq2 protocol to increase speed and accuracy. This protocol is useful for analyzing the pronounced heterogeneity of activation phenotypes in wound macrophages and might be adapted to other experimental models of skin inflammation. For complete details on the use and execution of this protocol, please refer to Willenborg et al. (2021).


Assuntos
Macrófagos , Cicatrização , Animais , Citometria de Fluxo , Contagem de Leucócitos , Camundongos , Análise de Sequência de RNA
3.
Front Immunol ; 13: 815828, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35493491

RESUMO

Mass cytometry has revolutionized immunophenotyping, particularly in exploratory settings where simultaneous breadth and depth of characterization of immune populations is needed with limited samples such as in preclinical and clinical tumor immunotherapy. Mass cytometry is also a powerful tool for single-cell immunological assays, especially for complex and simultaneous characterization of diverse intratumoral immune subsets or immunotherapeutic cell populations. Through the elimination of spectral overlap seen in optical flow cytometry by replacement of fluorescent labels with metal isotopes, mass cytometry allows, on average, robust analysis of 60 individual parameters simultaneously. This is, however, associated with significantly increased complexity in the design, execution, and interpretation of mass cytometry experiments. To address the key pitfalls associated with the fragmentation, complexity, and analysis of data in mass cytometry for immunologists who are novices to these techniques, we have developed a comprehensive resource guide. Included in this review are experiment and panel design, antibody conjugations, sample staining, sample acquisition, and data pre-processing and analysis. Where feasible multiple resources for the same process are compared, allowing researchers experienced in flow cytometry but with minimal mass cytometry expertise to develop a data-driven and streamlined project workflow. It is our hope that this manuscript will prove a useful resource for both beginning and advanced users of mass cytometry.


Assuntos
Anticorpos , Análise de Célula Única , Citometria de Fluxo/métodos , Imunofenotipagem , Análise de Célula Única/métodos , Coloração e Rotulagem
4.
J Vis Exp ; (182)2022 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-35532241

RESUMO

The conventional semen parameter analysis is widely used to assess male fertility. However, studies have found that ~15% of infertile patients show no abnormalities in conventional semen parameters. Additional technologies are needed to explain the idiopathic infertility and detect subtle sperm defects. Currently, biomarkers of sperm function, including sperm apoptosis, mitochondrial membrane potential (MMP), and DNA damage, reveal sperm physiology at the molecular level and are capable of predicting male fertility. With flow cytometry (FCM) techniques, each of these markers can be rapidly, accurately, and precisely measured in human semen samples, but time costs substantially increase and results could be obstructed if all the biomarkers need to be tested with a single cytometer. In this protocol, after collection and immediate incubation at 37 °C for liquefication, semen samples were further analyzed for sperm apoptosis using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. The MMP was labeled with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) probe, and DNA damage was assessed using the sperm chromatin structure assay (SCSA) with acridine orange (AO) staining. Thus, flow cytometric analysis of sperm function markers can be a practical and reliable toolkit for the diagnosis of infertility and evaluation of sperm function at both bench and bed.


Assuntos
Infertilidade , Espermatozoides , Biomarcadores , Cromatina , Citometria de Fluxo/métodos , Humanos , Masculino , Metaloproteinases da Matriz
5.
Methods Enzymol ; 667: 59-77, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35525555

RESUMO

Protein kinases catalyze the transfer of a phosphate group thereby activating proteins and initiating signaling cascades. Their cousins, the pseudokinases, are enzymatically nonactive counterparts of protein kinases that can be considered zombie enzymes. Interestingly, pseudokinases, which constitute about 10% of the human kinome, have been implicated in many cancers, despite their sequences predicting a lack of catalytic activity. Owing to recent research, it has been demonstrated that dysregulation of many pseudokinases triggers changes in cell signaling, proliferation, and drug resistance. This review is aimed at describing methods that can be used for detection of Tribbles family of pseudokinases, specifically TRIB2. We describe intracellular staining by flow cytometry and Western blotting techniques for the detection of endogenous TRIB2 protein.


Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina , Peptídeos e Proteínas de Sinalização Intracelular , Western Blotting , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Citometria de Fluxo , Humanos
6.
PLoS One ; 17(5): e0267911, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35522665

RESUMO

Molecular cloning, gene manipulation, gene expression, protein function, and gene regulation all depend on the introduction of nucleic acids into target cells. Multiple methods have been developed to facilitate such delivery including instrument based microinjection and electroporation, biological methods such as transduction, and chemical methods such as calcium phosphate precipitation, cationic polymers, and lipid based transfection, also known as lipofection. Here we report attempts to lipofect sea urchin coelomocytes using DOTAP lipofection reagent packaged with a range of molecules including fluorochromes, in addition to expression constructs, amplicons, and RNA encoding GFP. DOTAP has low cytotoxicity for coelomocytes, however, lipofection of a variety of molecules fails to produce any signature of success based on results from fluorescence microscopy and flow cytometry. While these results are negative, it is important to report failed attempts so that others conducting similar research do not repeat these approaches. Failure may be the outcome of elevated ionic strength of the coelomocyte culture medium, uptake and degradation of lipoplexes in the endosomal-lysosomal system, failure of the nucleic acids to escape the endosomal vesicles and enter the cytoplasm, and difficulties in lipofecting primary cultures of phagocytic cells. We encourage others to build on this report by using our information to optimize lipofection with a range of other approaches to work towards establishing a successful method of transfecting adult cells from marine invertebrates.


Assuntos
Ácidos Nucleicos , Ouriços-do-Mar , Animais , Cátions , Citometria de Fluxo , Lipossomos , Transfecção
7.
Front Immunol ; 13: 795815, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35493505

RESUMO

In the present work, we developed and evaluated the performance of a new flow cytometry-based single platform, referred to as "FC-Duplex IgG1 (HTLV-1/2)", for universal and differential serodiagnosis of HTLV-1/2 infection. The proposed technology employs a system for detection of IgG1 antibodies in a single competitive immunofluorescence platform by flow cytometry using fluorescently labeled MT-2/MoT cell line mix coupled to a highly sensitive development system (Biotin/Streptavidin/Phycoerythrin). The stability of fluorescent labeling and the antigenicity of MT-2 and MoT cell lines were confirmed upon storage at -20°C for 2, 6, and 12 months. The anti-HTLV-1/2 IgG1 reactivity, expressed as percentage of positive fluorescent cells (PPFC), was evaluated for each target antigen along the titration curve of test serum samples (1:32 to 1:4,096). Upon selection of target cell line and serum dilutions with higher segregation score between groups, the performance of "FIX" and "FIX & PERM" protocols was evaluated. The "FIX" protocol presented excellent performance indices (Se = 92%/Sp = 94%/AUC = 0.96; Se = 96%/Sp = 100%/AUC = 0.99) for the universal (HTLV-1/2 vs. NI) and differential (HTLV-1 vs. HTLV-2) diagnosis of HTLV-1 infection, respectively. Optimization of the "FIX" protocol using the principle of synchronous and asynchronous pairwise analysis further improved the performance of "FC-Duplex IgG1 (HTLV-1/2)", using the "FIX" protocol for differential diagnosis of HTLV-1 and HTLV-2 infections (Se = 100%/Sp = 100%/AUC = 1.00). In conclusion, the "FC-Duplex IgG1 (HTLV-1/2)" method represents an innovation in the biotechnology segment with the potential to compose a serological kit for differential diagnosis of HTLV-1/2 infection for reference laboratories and blood centers.


Assuntos
Infecções por HTLV-I , Vírus Linfotrópico T Tipo 1 Humano , Citometria de Fluxo/métodos , Infecções por HTLV-I/diagnóstico , Humanos , Imunoglobulina G , Testes Sorológicos
8.
Methods Mol Biol ; 2429: 345-356, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35507172

RESUMO

Müller glia (MG) are a relatively quiescent radial glial cell population capable of dedifferentiating to regenerate cells in the zebrafish retina that are lost due to damage. Here, we provide a protocol to both quantify MG cell dedifferentiation behavior during a regenerative response and isolate MG cells by fluorescence activated cell sorting (FACS). First, the retina is exposed to high-intensity light to induce retinal damage and either processed for immunohistochemistry or live MG cells are isolated by FACS that can be used for subsequent genomic or transcriptomic analyses. This method allows us to correlate MG cell behavior observed in situ with their transcriptomic profile at different stages during the regenerative response.


Assuntos
Células Ependimogliais , Peixe-Zebra , Animais , Proliferação de Células/fisiologia , Citometria de Fluxo , Larva , Neuroglia , Retina
9.
Mem Inst Oswaldo Cruz ; 117: e210157, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35508030

RESUMO

BACKGROUND: Leishmania parasites cause leishmaniasis that range from self-limiting cutaneous lesions to more serious forms of the disease. The search for potential drug targets focusing on biochemical and metabolic pathways revealed the sterol biosynthesis inhibitors (SBIs) as a promising approach. In this class of inhibitors is found ketoconazole, a classical inhibitor of 14α-methysterol 14-demethylase. OBJECTIVE: The present study aimed to better understand the biological response of Leishmania (Leishmania) amazonensis promastigotes at the cellular level after ketoconazole treatment. METHODS: Herein, techniques, such as fluorimetry, flow cytometry, fluorescence microscopy, electron and scanning microscopy were used to investigate the cellular structures and to identify organelles affected by ketoconazole treatment. FINDINGS: The study demonstrated, for the first time, the effect of ketoconazole on mitochondrion functioning and its probable relationship to cell cycle and death on L. (L.) amazonensis promastigotes (IFLA/BR/67/PH8 strain). MAIN CONCLUSIONS: Ketoconazole-induced mitochondrial damages led to hyperpolarisation of this single organelle and autophagic vacuoles formation, as a parasite survival strategy. These damages did not reflect directly on the parasite cell cycle, but drove the parasites to death, making them susceptible to ketoconazole treatment in in vitro models.


Assuntos
Antiprotozoários , Leishmania , Leishmaniose , Animais , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Citometria de Fluxo , Humanos , Cetoconazol/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias
10.
Radiol Technol ; 93(5): 454-461, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35508410

RESUMO

PURPOSE: To improve cardiovascular disease (CVD) risk prediction by combining screening techniques and to determine whether the combination of sonographic aortic calcification quantification, measurement of aortic intimal thickness, and monocyte laboratory values provides improved diagnostic detection compared with computed tomography (CT) calcium scoring. METHODS: A pre-experimental design was used to collect imaging, demographic, and biometric data. Data were collected from a convenience sample of 11 volunteers aged 40 to 60 years, including 6 men and 5 women. Collected data included anthropometric measures, laboratory values, flow cytometry, coronary artery calcium scores, atherosclerotic cardiovascular disease (ASCVD) 10-year risk scores, and aortic intimal-medial thickness (IMT). RESULTS: Aortic IMT in the distal portion of the aorta or region 1 was related significantly to mass (r = 0.725, P = .012), body mass index (r = 0.668, P = .025), and ASCVD 10-year risk score (r = 0.747, P = .033). The aortic IMT in mid portion of the aorta or region 2 was related significantly to mass (r = 0.651, P = .030), antihypertensive medications (r = 0.682, P = .021), ASCVD 10-year risk score (r = 0.753, P = .031), and total coronary artery calcification (CAC) (r = 0.626, P = .039). In addition, the proportions of circulating CD14+CD16- (traditional) and CD14+CD16+ (inflammatory) monocytes, and the monocyte surface expression of the adhesion molecules CD11a and CD11c, were correlated with the number of calcifications in regions 1 and 2. DISCUSSION: The use of a modified grading system for sonography provided a nonionizing, noninvasive option to easily assess patients' risks of CVD in an office environment. Although CAC has been used widely as a screening mechanism for CVD, ionizing radiation use might not be justified for those who are asymptomatic. The combination of sonography with flow cytometry demonstrated a promising alternative for assessing CVD risk. CONCLUSION: tBetter quantification of inflammatory markers and atherosclerotic plaques is needed. The combination of noninvasive imaging and advanced laboratory analysis holds promise for assessing and managing CVD risk. This study provides further evidence of the need for continued research with larger sample sizes and diversified populations to improve the quality of CVD risk assessment.


Assuntos
Aterosclerose , Calcinose , Doenças Cardiovasculares , Doença da Artéria Coronariana , Aorta/diagnóstico por imagem , Calcinose/diagnóstico por imagem , Cálcio , Doença da Artéria Coronariana/diagnóstico por imagem , Feminino , Citometria de Fluxo , Humanos , Masculino , Projetos Piloto , Medição de Risco , Fatores de Risco , Tomografia Computadorizada por Raios X
11.
Methods Mol Biol ; 2469: 193-200, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35508840

RESUMO

Plant organs are built of different cell types, characterized by specific transcription programs and metabolic profiles. The possibility of isolation of such cell types to perform differential transcriptomic, proteomic and metabolomic analyses is highly important to understand many aspects of plant physiology, namely, the structure and regulation of economically valuable specialized metabolic pathways. Here, we describe the isolation of idioblast leaf protoplasts of the medicinal plant Catharanthus roseus by fluorescence-activated cell sorting, taking advantage of the differential autofluorescence properties of those specialized cells.


Assuntos
Catharanthus , Células Vegetais , Citometria de Fluxo , Regulação da Expressão Gênica de Plantas , Células Vegetais/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Proteômica
12.
Front Immunol ; 13: 805420, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35359928

RESUMO

The interstitium of kidney involves a variety of components including resident immune cells, in particular mononuclear phagocytes. However, many proposed markers for distinguishing macrophages or dendritic cells are, in fact, shared by the majority of renal mononuclear phagocytes, which impedes the research of kidney diseases. Here, by employing a flow cytometry strategy and techniques of fate mapping, imaging and lineage depletion, we were able to demarcate renal monocytes, macrophages and dendritic cells and their subsets in mice. In particular, using this strategy, we found that even in steady state, the renal macrophage pool was continuously replenished by bone marrow-derived monocytes in a stepwise process, i.e., from infiltration of classical monocyte, to development of nonclassical monocyte and eventually to differentiation to macrophages. In mechanism, we demonstrated that the ligation of tissue-anchored CX3CL1 and monocytic CX3CR1 was required for promoting monocyte differentiation to macrophages in the kidney, but CX3CL1-CX3CR1 signaling was dispensable in monocyte infiltrating into the kidney. In addition to the bone marrow-derived macrophages, fate mapping in adult mice revealed another population of renal resident macrophages which were embryo-derived and self-maintaining. Thus, the dissecting strategies developed by us would assist in exploration of the biology of renal mononuclear phagocytes.


Assuntos
Macrófagos , Monócitos , Animais , Citometria de Fluxo , Rim , Contagem de Leucócitos , Camundongos
13.
Front Immunol ; 13: 867016, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35419006

RESUMO

Assessing the health and competence of the immune system is central to evaluating vaccination responses, autoimmune conditions, cancer prognosis, and treatment. With an increasing number of studies examining immune dysregulation, there is a growing need for a curated reference of variation in immune parameters in healthy individuals. We used mass cytometry (CyTOF) to profile blood from 86 humans in response to 15 ex vivo immune stimuli. We present reference ranges for cell-specific immune markers and highlight differences that appear across sex and age. We identified modules of immune features that suggest there exists an underlying structure to the immune system based on signaling pathway responses across cell types. We observed increased MAPK signaling in inflammatory pathways in innate immune cells and greater overall coordination of immune cell responses in females. In contrast, males exhibited stronger pSTAT1 and pTBK1 responses. These reference data are publicly available as a resource for immune profiling studies.


Assuntos
Doenças Autoimunes , Transdução de Sinais , Biomarcadores , Feminino , Citometria de Fluxo , Humanos , Sistema Imunitário , Masculino
14.
Anal Chem ; 94(15): 5769-5775, 2022 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-35384647

RESUMO

In order to obtain high yield of astaxanthin, a high-value compound with ultrastrong antioxidant capacity, it is necessary to identify the growth characteristics (biomass, morphology, and size) of Haematococcus pluvialis. The current detection methods have the disadvantages of labor-consuming operation or complicated measurement system. It is an urgent need to explore a simple and cost-effective method for the detection of H. pluvialis with large size distribution during its growth period. In this work, a digital in-line holographic flow cytometry using a linear array sensor is proposed to measure the growth characteristics of H. pluvialis in a two-dimensional (2-D) hydrodynamic focusing microfluidic chip. Based on the modified angular spectrum method, the distorting holograms caused by the asynchrony of sample flow velocity and acquisition speed of the linear array sensor were rectified and reconstructed. In addition, the depth-of-focus of the imaging system were digitally extended to cover the entire depth of the microfluidic channel for optimized imaging quality. We have utilized the proposed method to statistically investigate the biomass, morphology and size of H. pluvialis under different culture conditions and growth durations.


Assuntos
Clorofíceas , Microfluídica , Antioxidantes , Biomassa , Citometria de Fluxo
15.
J Immunol Res ; 2022: 8720438, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35437510

RESUMO

Severe pneumonia accounts for the majority of morbidity and mortality in renal allograft recipients due to immunosuppressant maintenance. Regulatory T cells (Tregs), which are involved in tackling infections under immunosuppressive conditions, are rarely uncovered. We aimed to investigate the relationship between various Treg subpopulations and severe pneumonia after kidney transplantation (KTx). KTx recipients with pneumonia were divided into severe pneumonia and mild pneumonia groups. The frequencies and absolute numbers (Ab No.) of total Tregs (CD4+CD25+FoxP3+), six subsets of Tregs (Helios+/-, CD39+/-, and CD45RA+/-), and T cells, B cells, and NK cells were assessed from peripheral blood via flow cytometry using the t or Mann-Whitney test and receiver operating curve analysis. We also determined the median fluorescence intensity (MFI) of human leukocyte antigen- (HLA-) DR on monocytes and CD64 on neutrophils. Logistic regression was used to identify the risk factors of disease progression, and Pearson's correlation analysis was performed to identify relationships between the measured immune indices and patients' clinical information. Our research indicated that Treg subpopulations were strongly associated with severe pneumonia progression post KTx. Based on the monitoring of Treg subpopulations, better-individualized prevention and therapy might be achieved for patients with severe pneumonia post KTx.


Assuntos
Transplante de Rim , Pneumonia , Citometria de Fluxo , Fatores de Transcrição Forkhead , Antígenos HLA , Humanos , Imunossupressores , Transplante de Rim/efeitos adversos , Pneumonia/etiologia , Linfócitos T Reguladores
16.
ACS Appl Mater Interfaces ; 14(15): 17128-17141, 2022 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-35385643

RESUMO

Hybridoma technology is widely used for monoclonal antibody (mAb) discovery, whereas the generation and identification of single hybridomas by the limiting dilution method (LDM) are tedious, inefficient, and time- and cost-consuming, especially for hapten molecules. Here, we describe a single transgenic hybridoma selection method (STHSM) that employs a transgenic Sp2/0 with an artificial and stable on-cell-surface anchor. The anchor was designed by combining the truncated variant transmembrane domain of EGFR with a biotin acceptor peptide AVI-tag, which was stably integrated into the genome of Sp2/0 via a piggyBac transposon. To ensure the subsequent precise selection of the hybridoma, the number of on-cell-surface anchors of the transfected Sp2/0 for fusion with immunized splenocytes was further normalized by flow cytometry at the single cell level. Then the single antigen-specific transgenic hybridomas were precisely identified and automatically selected using a CellenONE platform based on the fluorescence assay of the on-cell-surface anchor with the corresponding secreted antigen-specific mAb. The STHSM produced 579 single chloramphenicol (CAP)-specific transgenic hybridomas with a positive rate of 62.7% in 10 plates within 2 h by one-step selection, while only 12 single CAP-specific hybridomas with a positive rate of 6.3% in 40 plates required at least 32 days using the LDM with multiple subcloning steps. The best affinity of mAbs from the STHSM was more than 2-fold higher than that of those from the LDM, and this was mainly due to the preaffinity selection based on the on-cell-surface anchors and more interactions between the mAb and CAP. Then the mAbs from the STHSM and LDM were used to develop an immunoassay for CAP in spiked and natural biological samples. The method displayed satisfactory sensitivity, accuracy, and precision, demonstrating that the STHSM we developed is a versatile, practical, and efficient method for mAb discovery.


Assuntos
Anticorpos Monoclonais , Antígenos , Animais , Anticorpos Monoclonais/genética , Citometria de Fluxo , Haptenos , Hibridomas , Camundongos , Camundongos Endogâmicos BALB C
17.
Methods Mol Biol ; 2491: 29-62, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35482183

RESUMO

Yeast surface display is a powerful directed evolution method for developing and engineering protein molecules to attain desired properties. Here, updated protocols are presented for purposes of identification of lead binders and their affinity maturation. Large libraries are screened by magnetic bead selections followed by flow cytometric selections. Upon identification and characterization of single clones, their affinities are improved by an iterative process of mutagenesis and fluorescence-activated cell sorting.


Assuntos
Engenharia de Proteínas , Saccharomyces cerevisiae , Citometria de Fluxo/métodos , Biblioteca Gênica , Mutagênese , Engenharia de Proteínas/métodos , Saccharomyces cerevisiae/genética
18.
Methods Mol Biol ; 2491: 105-115, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35482187

RESUMO

Yeast surface display is a robust platform for obtaining binders with high affinity. Kinetic competition screening is an effective method for maturing the affinity of binders with strong starting affinities, or when dissociation kinetics are a key consideration for the protein of interest. In this chapter, we describe detailed protocols for setting up and performing a kinetic competition screen. The duration of competition is determined based on the dissociation rate constant of the parental clone measured on the yeast surface. This methodology was successfully used to improve the affinity of a viral double-stranded RNA binding protein with starting affinity in the sub-nanomolar range.


Assuntos
Pesquisa , Saccharomyces cerevisiae , Citometria de Fluxo/métodos , Cinética , Proteínas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
19.
Methods Mol Biol ; 2491: 177-193, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35482191

RESUMO

Yeast surface display (YSD) emerged as a prominent screening methodology for the isolation of monoclonal antibodies (mAbs) against various antigens. However, phage display remains the gold standard in cell panning-based screenings to isolate mAbs against difficult-to-screen targets, such as G-protein coupled receptors (GPCR) and ion channels. Herein we describe a step-by-step protocol to establish and perform the isolation of mAbs using YSD in a fluorescence-activated cell sorting (FACS)-assisted biopanning manner, yielding a variety of antibodies binding their antigen with high affinity in the natural environment of the cell. Upon mixing antibody-displaying yeast cells with antigen-displaying mammalian cells, complexes are specifically formed and isolated for enrichment of yeast cells encoding binders against the antigen. The utilization of mammalian cells expressing the respective target accounts for accessibility of the epitope and the correct conformation of the antigen. Furthermore, critical characterization methods mandatory for this kind of antibodies are illuminated.


Assuntos
Bioprospecção , Saccharomyces cerevisiae , Animais , Anticorpos Monoclonais/química , Antígenos , Técnicas de Visualização da Superfície Celular , Citometria de Fluxo/métodos , Mamíferos , Biblioteca de Peptídeos , Saccharomyces cerevisiae/metabolismo
20.
Methods Mol Biol ; 2491: 195-216, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35482192

RESUMO

Membrane proteins are favored drug targets and antibody therapeutics represent the fastest-growing category of pharmaceuticals. However, there remains a need for rapid and effective approaches for the discovery of antibodies that recognize membrane proteins to develop a robust clinical pipeline for targeted therapeutics. The challenges associated with recombinant expression of membrane proteins make whole cell screening techniques desirable, as these strategies allow presentation of the target membrane proteins in their native conformations. Here, we describe a workflow that employs both adherent cell-based and suspension cell-based whole cell panning methodologies to enrich for specific binders within a yeast-displayed antibody library. The first round of selection consists of an adherent cell-based approach, wherein a diverse library is panned over target-expressing mammalian cell monolayers in order to debulk the naïve library. Subsequent rounds involve the use of suspension cell-based approaches, facilitated with magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS), to achieve further library enrichment. Finally, we describe a high-throughput approach to screen target binding and specificity of individual clones isolated from selection campaigns.


Assuntos
Proteínas de Membrana , Biblioteca de Peptídeos , Animais , Anticorpos , Citometria de Fluxo/métodos , Testes Imunológicos , Mamíferos , Proteínas de Membrana/genética , Suspensões
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