Journal of biological methods最新文献_第9页

A 2D and 3D melanogenesis model with human primary cells induced by tyrosine. 酪氨酸诱导人原代细胞的二维和三维黑色素生成模型。

Journal of biological methods Pub Date : 2020-07-02 eCollection Date: 2020-01-01 DOI: 10.14440/jbm.2020.327

Maryana S Branquinho, Maysa B Silva, Jacqueline C Silva, Maria C Sales, Silvia B Barros, Silvya S Maria-Engler, Ana Campa

{"title":"A 2D and 3D melanogenesis model with human primary cells induced by tyrosine.","authors":"Maryana S Branquinho, Maysa B Silva, Jacqueline C Silva, Maria C Sales, Silvia B Barros, Silvya S Maria-Engler, Ana Campa","doi":"10.14440/jbm.2020.327","DOIUrl":"https://doi.org/10.14440/jbm.2020.327","url":null,"abstract":"Research on melanogenesis, its regulation in health and disease, and the discovery of new molecules with pigmenting and depigmenting activities use different models. Here we standardize a protocol based on previous ones using primary human melanocytes and keratinocytes in co-cultures, in which melanogenesis was induced under mild conditions by the addition of tyrosine plus ammonium chloride (NH4Cl). The expression of MITF, TYR, TYRP1, and Melan-A as well as melanin content were measured. Furthermore, we extended this study to a reconstructed 3D model. Pigmentation was visually observable and melanosomes were identified by Fontana-Masson staining by the addition of tyrosine plus NH4Cl during the stratification phase. The 2D and 3D protocols proposed here circumvent limitations of previous models, using human primary cells and mild conditions for melanogenesis. These protocols offer a viable, robust, simple, and animal-free investigational option for human skin pigmentation studies and screening tests for new compounds that modulate pigmentation.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"7 3","pages":"e134"},"PeriodicalIF":0.0,"publicationDate":"2020-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/ec/73/jbm-7-3-e134.PMC7483082.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38385286","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 3

Real time visualization of cancer cell death, survival and proliferation using fluorochrome-transfected cells in an IncuCyte^® imaging system. 在IncuCyte®成像系统中使用荧光染料转染的细胞实时可视化癌细胞死亡，存活和增殖。

Journal of biological methods Pub Date : 2020-06-12 eCollection Date: 2020-01-01 DOI: 10.14440/jbm.2020.323

Thomas M Lanigan, Stephanie M Rasmussen, Daniel P Weber, Kalana S Athukorala, Phillip L Campbell, David A Fox, Jeffrey H Ruth

{"title":"Real time visualization of cancer cell death, survival and proliferation using fluorochrome-transfected cells in an IncuCyte® imaging system.","authors":"Thomas M Lanigan, Stephanie M Rasmussen, Daniel P Weber, Kalana S Athukorala, Phillip L Campbell, David A Fox, Jeffrey H Ruth","doi":"10.14440/jbm.2020.323","DOIUrl":"https://doi.org/10.14440/jbm.2020.323","url":null,"abstract":"Cancer immunotherapy is a rapidly advancing and viable approach to treating cancer along with more traditional forms of therapy. Real-time cell analysis technologies that examine the dynamic interactions between cancer cells and the cells of the immune system are becoming more important for assessment of novel therapeutics. In this report, we use the IncuCyte® imaging system to study the killing potential of various immune cells on cancer cell lines. The IncuCyte® system tracks living cells, labeled by a red fluorescent protein, and cell death, as indicated by the caspase-3/7 reagent, which generates a green fluorescent signal upon activation of apoptotic pathways. Despite the power of this approach, obtaining commercially fluorescent cancer cell lines is expensive and limited in the range of cell lines that are available. To overcome this barrier, we developed an inexpensive method using a lentiviral construct expressing nuclear localized mKate2 red fluorescent protein to stably label cancer cells. We demonstrate that this method is effective in labeling a wide variety of cell lines, allowing for analyses of different cancers as well as different cell lines of the same type of cancer.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"7 2","pages":"e133"},"PeriodicalIF":0.0,"publicationDate":"2020-06-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/b6/35/jbm-7-2-e133.PMC7300428.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38079873","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 7

A simple method for long-term vital-staining of ciliated epidermal cells in aquatic larvae. 水生幼虫纤毛表皮细胞长期活体染色的简单方法。

Journal of biological methods Pub Date : 2020-04-29 eCollection Date: 2020-01-01 DOI: 10.14440/jbm.2020.320

Jörn von Döhren, Sabrina Kuhl

{"title":"A simple method for long-term vital-staining of ciliated epidermal cells in aquatic larvae.","authors":"Jörn von Döhren, Sabrina Kuhl","doi":"10.14440/jbm.2020.320","DOIUrl":"https://doi.org/10.14440/jbm.2020.320","url":null,"abstract":"Observing the process of growth and differentiation of tissues and organs is of crucial importance for the understanding of the evolution of organs in animals. Unfortunately, it is notoriously difficult to continuously monitor developmental processes due to the extended time they take. Long-term labeling of the tissues of interest represents a promising alternative to raise these pivotal data. In the case of the prototroch, a band of ciliated cells typical of marine, planktotrophic trochophora larvae, we were able to apply a long-term fluorescent vital-staining to the prototroch cells that remains detectable throughout further larval life. We were able to stain ciliated cells of planktonic larvae from different spiralian clades by using long-chain dialkylcarbocyanine dyes that are detectable in different fluorescent emission spectra in combination with a non-ionic surfactant. The larvae survived and developed normally, their ciliated cells retaining the originally applied fluorescent labels. Combined with additional fluorescent staining of the larvae after fixation, we provide an easy, versatile, and broadly applicable method to investigate the processes of the differentiation of epidermal organs in various aquatic larvae.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"7 2","pages":"e132"},"PeriodicalIF":0.0,"publicationDate":"2020-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/7c/7d/jbm-7-2-e132.PMC7300427.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38079872","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Standardized 11-color flow cytometry panel for the functional phenotyping of human T regulatory cells. 标准化的11色流式细胞仪面板用于人类T调节细胞的功能表型。

Journal of biological methods Pub Date : 2020-04-13 eCollection Date: 2020-01-01 DOI: 10.14440/jbm.2020.325

Claire Manuszak, Martha Brainard, Emily Thrash, F Stephen Hodi, Mariano Severgnini

引用次数: 6

A system for the high-throughput analysis of acute thermal avoidance and adaptation in C. elegans. 秀丽隐杆线虫急性热回避和适应的高通量分析系统。

Journal of biological methods Pub Date : 2020-03-17 eCollection Date: 2020-01-01 DOI: 10.14440/jbm.2020.324

Andrei-Stefan Lia, Dominique A Glauser

{"title":"A system for the high-throughput analysis of acute thermal avoidance and adaptation in C. elegans.","authors":"Andrei-Stefan Lia, Dominique A Glauser","doi":"10.14440/jbm.2020.324","DOIUrl":"https://doi.org/10.14440/jbm.2020.324","url":null,"abstract":"Nociception and its plasticity are essential biological processes controlling adaptive behavioral responses in animals. These processes are also linked to different pain conditions in human and have received considerable attention, notably via studies in rodent models and the use of heat-evoked withdrawal behavior assays as a readout of unpleasant experience. More recently, invertebrates have also emerged as useful complementary models, with their own set of advantages, including their amenability to genetic manipulations, the accessibility and relative simplicity of their nervous system and ethical concerns linked to animal suffering. Like humans, the nematode Caenorhabditis elegans (C. elegans) can detect noxious heat and produce avoidance responses such as reversals. Here, we present a methodology suitable for the high-throughput analysis of C. elegans heat-evoked reversals and the adaptation to repeated stimuli. We introduce two platforms: the INFERNO (for infrared-evoked reversal analysis platform), allowing the quantification of the thermal sensitivity in a petri dish containing a large population (> 100 animals), and the ThermINATOR (for thermal adaptation multiplexed induction platform), allowing the mass-adaptation of up to 18 worm populations at the same time. We show that wild type animals progressively desensitize in response to repeated noxious heat pulses. Furthermore, analyzing the phenotype of mutant animals, we show that the mechanisms underlying baseline sensitivity and adaptation, respectively, are supported by genetically separable molecular pathways. In conclusion, the presented method enables the high-throughput evaluation of thermal avoidance in C. elegans and will contribute to accelerate studies in the field with this invertebrate model.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"7 1","pages":"e129"},"PeriodicalIF":0.0,"publicationDate":"2020-03-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/ab/26/jbm-7-1-e129.PMC7163209.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37853979","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 3

Optimized methodology for product recovery following emulsion PCR: applications for amplification of aptamer libraries and other complex templates. 乳液 PCR 后产品回收的优化方法：应用于扩增适配体文库和其他复杂模板。

Journal of biological methods Pub Date : 2020-03-10 eCollection Date: 2020-01-01 DOI: 10.14440/jbm.2020.316

Grant C O'Connell, Christine G Smothers

{"title":"Optimized methodology for product recovery following emulsion PCR: applications for amplification of aptamer libraries and other complex templates.","authors":"Grant C O'Connell, Christine G Smothers","doi":"10.14440/jbm.2020.316","DOIUrl":"10.14440/jbm.2020.316","url":null,"abstract":"Bias and background issues make efficient amplification of complex template mixes such as aptamer and genomic DNA libraries via conventional PCR methods difficult; emulsion PCR is being increasingly used in such scenarios to circumvent these problems. However, before products generated via emulsion PCR can be used in downstream workflows, they need to be recovered from the water-in-oil emulsion. Often, emulsions are broken following amplification using volatile organic solvents, and product is subsequently isolated via precipitation. Unfortunately, the use of such solvents requires the implementation of special environmental controls, and the yield and purity of DNA isolated by precipitation can be highly variable. Here, we describe the optimization of a simple protocol which can be used to recover products following emulsion PCR using a 2-butanol extraction and subsequent DNA isolation via a commercially available clean-up kit. This protocol avoids the use of volatile solvents and precipitation steps, and we demonstrate that it can be used to reliably recover DNA from water-in-oil emulsions with efficiencies as high as 90%. Furthermore, we illustrate the practical applicability of this protocol by demonstrating how it can be implemented to recover a complex random aptamer library following amplification via emulsion PCR.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"7 1","pages":"e128"},"PeriodicalIF":0.0,"publicationDate":"2020-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/89/3a/jbm-7-1-e128.PMC7081054.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37762929","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

An optimized procedure for isolation of rodent and human skeletal muscle sarcoplasmic and myofibrillar proteins. 分离啮齿动物和人类骨骼肌肌浆蛋白和肌原纤维蛋白的优化程序。

Journal of biological methods Pub Date : 2020-02-24 eCollection Date: 2020-01-01 DOI: 10.14440/jbm.2020.307

Michael D Roberts, Kaelin C Young, Carlton D Fox, Christopher G Vann, Paul A Roberson, Shelby C Osburn, Johnathon H Moore, Petey W Mumford, Matthew A Romero, Darren T Beck, Cody T Haun, Veera L D Badisa, Benjamin M Mwashote, Victor Ibeanusi, Andreas N Kavazis

引用次数: 16

Generation of bone marrow chimeras using X-ray irradiation: comparison to cesium irradiation and use in immunotherapy. x射线照射产生骨髓嵌合体:与铯照射的比较及其在免疫治疗中的应用。

Journal of biological methods Pub Date : 2020-02-17 eCollection Date: 2020-01-01 DOI: 10.14440/jbm.2020.314

Jason Eng, Jessica Orf, Kristy Perez, Deepali Sawant, Jason DeVoss

{"title":"Generation of bone marrow chimeras using X-ray irradiation: comparison to cesium irradiation and use in immunotherapy.","authors":"Jason Eng, Jessica Orf, Kristy Perez, Deepali Sawant, Jason DeVoss","doi":"10.14440/jbm.2020.314","DOIUrl":"https://doi.org/10.14440/jbm.2020.314","url":null,"abstract":"Bone marrow chimeras represent a key tool employed to understand biological contributions stemming from the hematopoietic versus the stromal compartment. In most institutions, cesium irradiators are used to lethally irradiate recipient animals prior to the injection of donor bone marrow. Cesium irradiators, however, have significant liabilities—including concerns around domestic security. Recently, X-ray irradiators have been implemented as a potential alternative to cesium sources. Only a small number of publications in the literature have attempted to compare these two modalities and, in most cases, the emphasis was on irradiation of human blood productions. We were able to find only a single study that directly compared X-ray and cesium technologies in the generation of murine bone marrow chimeras, a standard laboratory practice. This study focused on chimerism in the blood of recipient animals. In the present study, we begin by comparing cesium and X-ray based sources for irradiation, then transition to using X-ray-based systems for immunology models with an emphasis on immunotherapy of cancer in immunocompetent mouse models—specifically evaluating chimerism in the blood, spleen, and tumor microenvironment. While our data demonstrate that the two platforms are functionally comparable and suggest that X-ray based technology is a suitable alternative to cesium sources. We also highlight a difference in chimerism between the peripheral (blood, spleen) and tumor compartments that is observed using both technologies. While the overall degree of chimerism in the peripheral tissues is very high, the degree of chimerism in the tumor is cell type specific with T and NK cells showing lower chimerism than other cell types.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"7 1","pages":"e125"},"PeriodicalIF":0.0,"publicationDate":"2020-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/94/4f/jbm-7-1-e125.PMC7082502.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37766013","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 7

A simple method for short-term maintenance of neonatal mice without foster mothers. 无养母新生小鼠短期维持的简单方法。

Journal of biological methods Pub Date : 2020-02-17 eCollection Date: 2020-01-01 DOI: 10.14440/jbm.2020.315

Craig Kreikemeier-Bower, Pascal Polepole, Katherine Pinkerton, Luwen Zhang

引用次数: 1

Highly efficient induced pluripotent stem cell reprogramming of cryopreserved lymphoblastoid cell lines. 低温保存淋巴母细胞样细胞系的高效诱导多能干细胞重编程。

Journal of biological methods Pub Date : 2020-01-08 eCollection Date: 2020-01-01 DOI: 10.14440/jbm.2020.296

Satish Kumar, Joanne E Curran, Erika C Espinosa, David C Glahn, John Blangero

{"title":"Highly efficient induced pluripotent stem cell reprogramming of cryopreserved lymphoblastoid cell lines.","authors":"Satish Kumar, Joanne E Curran, Erika C Espinosa, David C Glahn, John Blangero","doi":"10.14440/jbm.2020.296","DOIUrl":"https://doi.org/10.14440/jbm.2020.296","url":null,"abstract":"Tissue culture based in-vitro experimental modeling of human inherited disorders provides insight into the cellular and molecular mechanisms involved and the underlying genetic component influencing the disease phenotype. The breakthrough development of induced pluripotent stem cell (iPSC) technology represents a quantum leap in experimental modeling of human diseases, providing investigators with a self-renewing and thus unlimited source of pluripotent cells for targeted differentiation into functionally relevant disease specific tissue/cell types. The existing rich bio-resource of Epstein-Barr virus (EBV) immortalized lymphoblastoid cell line (LCL) repositories generated from a wide array of patients in genetic and epidemiological studies worldwide, many of them with extensive genotypic, genomic and phenotypic data already existing, provides a great opportunity to reprogram iPSCs from any of these LCL donors in the context of their own genetic identity for disease modeling and disease gene identification. However, due to the low reprogramming efficiency and poor success rate of LCL to iPSC reprogramming, these LCL resources remain severely underused for this purpose. Here, we detailed step-by-step instructions to perform our highly efficient LCL-to-iPSC reprogramming protocol using EBNA1/OriP episomal plasmids encoding pluripotency transcription factors (i.e., OCT3/4, SOX2, KLF4, L-MYC, and LIN28), mouse p53DD (p53 carboxy-terminal dominant-negative fragment) and commercially available reprogramming media. We achieved a consistently high reprogramming efficiency and 100% success rate (> 200 reprogrammed iPSC lines) using this protocol.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"7 1","pages":"e124"},"PeriodicalIF":0.0,"publicationDate":"2020-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/f7/93/jbm-7-1-e124.PMC6974695.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37575406","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 8