Journal of biological methods最新文献_第8页

High-yield purification of exceptional-quality, single-molecule DNA substrates. 优质单分子DNA底物的高产量纯化。

Journal of biological methods Pub Date : 2021-02-24 eCollection Date: 2021-01-01 DOI: 10.14440/jbm.2021.350

Yue Lu, Piero Bianco

{"title":"High-yield purification of exceptional-quality, single-molecule DNA substrates.","authors":"Yue Lu, Piero Bianco","doi":"10.14440/jbm.2021.350","DOIUrl":"https://doi.org/10.14440/jbm.2021.350","url":null,"abstract":"Single-molecule studies involving DNA or RNA, require homogeneous preparations of nucleic acid substrates of exceptional quality. Over the past several years, a variety of methods have been published describing different purification methods but these are frustratingly inconsistent with variable yields even in the hands of experienced bench scientists. To address these issues, we present an optimized and straightforward, column-based approach that is reproducible and produces high yields of substrates or substrate components of exceptional quality. Central to the success of the method presented is the use of a non-porous anion exchange resin. In addition to the use of this resin, we encourage the optimization of each step in the construction of substrates. The fully optimized method produces high yields of a hairpin DNA substrate of exceptional quality. While this substrate is suitable for single-molecule, magnetic tweezer experiments, the described method is readily adaptable to the production of DNA substrates for the majority of single-molecule studies involving nucleic acids ranging in size from 70-15000 bp.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"8 1","pages":"e145"},"PeriodicalIF":0.0,"publicationDate":"2021-02-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/61/cd/jbm-8-1-e145.PMC8054919.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38901482","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: 1

Using a variant of the optomotor response as a visual defect detection assay in zebrafish. 使用视运动反应的变体作为斑马鱼的视觉缺陷检测试验。

Journal of biological methods Pub Date : 2021-02-01 eCollection Date: 2021-01-01 DOI: 10.14440/jbm.2021.341

Matthew K LeFauve, Cassie J Rowe, Mikayla Crowley-Perry, Jenna L Wiegand, Arthur G Shapiro, Victoria P Connaughton

{"title":"Using a variant of the optomotor response as a visual defect detection assay in zebrafish.","authors":"Matthew K LeFauve, Cassie J Rowe, Mikayla Crowley-Perry, Jenna L Wiegand, Arthur G Shapiro, Victoria P Connaughton","doi":"10.14440/jbm.2021.341","DOIUrl":"https://doi.org/10.14440/jbm.2021.341","url":null,"abstract":"We describe a visual stimulus that can be used with both larval and adult zebrafish (Danio rerio). This protocol is a modification of a standard visual behavior analysis, the optomotor response (OMR). The OMR is often used to determine the spatial response or to detect directional visuomotor deficiencies. An OMR can be generated using a high contrast grated pattern, typically vertical bars. The spatial sensitivity is measured by detection and response to a change in grating bar width and is reported in cycles per degree (CPD). This test has been used extensively with zebrafish larvae and adults to identify visual- and/or motor-based mutations. Historically, when tested in adults, the grated pattern was presented from a vertical perspective, using a rotating cylinder around a holding tank, allowing the grating to be seen solely from the sides and front of the organism. In contrast, OMRs in zebrafish larvae are elicited using a stimulus projected below the fish. This difference in methodology means that two different experimental set-ups are required: one for adults and one for larvae. Our visual stimulus modifies the stimulation format so that a single OMR stimulus, suitable for use with both adults and larvae, is being presented underneath the fish. Analysis of visuomotor responses using this method does not require costly behavioral tracking software and, using a single behavioral paradigm, allows the observer to rapidly determine visual spatial response in both zebrafish larvae and adults.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"8 1","pages":"e144"},"PeriodicalIF":0.0,"publicationDate":"2021-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/79/6d/jbm-8-1-e144.PMC7884848.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25383021","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: 9

Optimization of small-scale sample preparation for high-throughput OpenArray analysis. 用于高通量OpenArray分析的小规模样品制备优化。

Journal of biological methods Pub Date : 2021-02-01 eCollection Date: 2021-01-01 DOI: 10.14440/jbm.2021.339

Neeta A Abraham, Anne C Campbell, Warren D Hirst, Catherine L Nezich

{"title":"Optimization of small-scale sample preparation for high-throughput OpenArray analysis.","authors":"Neeta A Abraham, Anne C Campbell, Warren D Hirst, Catherine L Nezich","doi":"10.14440/jbm.2021.339","DOIUrl":"https://doi.org/10.14440/jbm.2021.339","url":null,"abstract":"OpenArray is one of the most high-throughput qPCR platforms available but its efficiency can be limited by sample preparation methods that are slow and costly. To optimize the sample workflow for high-throughput qPCR processing by OpenArray, small-scale sample preparation methods were compared for compatibility with this system to build confidence in a method that maintains quality and accuracy while using less starting material and saving time and money. This study is the first to show that the Cells-to-CT kit can be used to prepare samples within the dynamic range of OpenArray directly from cultured cells in a single well of a 96-well plate when used together with a cDNA preamplification PCR step. Use of Cells-to-CT produced results of similar quality and accuracy to that of a preparation method using purified RNA in less than half the sample preparation time. While Cells-to-CT samples also exhibited slightly increased variance, which affects the ability of OpenArray to distinguish small differences in gene expression, overall gene expression mean results correlated well between small-scale methods. This work demonstrates that Cells-to-CT with preamplification can be used to reliably prepare samples for OpenArray analysis while saving time, money, and starting material.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"8 1","pages":"e143"},"PeriodicalIF":0.0,"publicationDate":"2021-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/3e/51/jbm-8-1-e143.PMC7884849.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25382534","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

Visualization of subdiffusive sites in a live single cell. 活单细胞亚扩散部位的可视化。

Journal of biological methods Pub Date : 2021-01-30 eCollection Date: 2021-01-01 DOI: 10.14440/jbm.2021.348

Zeno Földes-Papp, Gerd Baumann, Long-Cheng Li

引用次数: 0

Comparison of different clearing and acquisition methods for 3D imaging of murine intestinal organoids. 小鼠肠道类器官三维成像不同清除获取方法的比较。

Journal of biological methods Pub Date : 2020-12-28 eCollection Date: 2020-01-01 DOI: 10.14440/jbm.2020.334

Louison Lallemant, Corinne Lebreton, Meriem Garfa-Traoré

引用次数: 5

A streamlined CRISPR/Cas9 approach for fast genome editing in Toxoplasma gondii and Besnoitia besnoiti. 一种用于刚地弓形虫和刚地弓形虫快速基因组编辑的简化CRISPR/Cas9方法

Journal of biological methods Pub Date : 2020-12-19 eCollection Date: 2020-01-01 DOI: 10.14440/jbm.2020.343

Rahel R Winiger, Adrian B Hehl

{"title":"A streamlined CRISPR/Cas9 approach for fast genome editing in Toxoplasma gondii and Besnoitia besnoiti.","authors":"Rahel R Winiger, Adrian B Hehl","doi":"10.14440/jbm.2020.343","DOIUrl":"https://doi.org/10.14440/jbm.2020.343","url":null,"abstract":"Toxoplasma gondii (T. gondii) and Besnoitia besnoiti (B. besnoiti) are closely related coccidian parasites belonging to the phylum Apicomplexa, which comprises many other important pathogens of humans and livestock. T. gondii is considered a model organism for studying the cell biology of Apicomplexa mainly due to the ease of propagation in diverse host cells and the availability of a wide range of genetic tools. Conversely, B. besnoiti in vitro culture systems currently exist only for the acute phase of infection, and genetic manipulation has proven much more challenging. In recent years, the targeted editing of chromosomal DNA by the programmable CRISPR-associated (Cas)9 enzyme has greatly improved the scope and accuracy of genetic manipulation in T. gondii and related parasites but is still lagging in B. besnoiti. The CRISPR/Cas9 technology enables the introduction of single point and insertion/deletion mutations, precise integration of in-frame epitope tags, and deletions of genes at reduced time and cost compared to previous methods. Current protocols for CRISPR-mediated genome editing in T. gondii rely on either constitutive or transient expression of Cas9 as well as target specific sgRNAs encoded separately or together on transfected plasmid vectors. Constitutively expressed Cas9 carries the risk of toxicity, whilst the transient approach is laborious and error-prone. Here we present a protocol for plasmid vector-independent genome-editing using chemically synthesized and modified sgRNAs. This protocol allows for rapid and cost-effective generation of mutant cell lines of T. gondii and B. besnoiti.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":" ","pages":"e140"},"PeriodicalIF":0.0,"publicationDate":"2020-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/06/2f/jbm-7-4-e140.PMC7865079.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25351451","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: 4

An adapted novel flow cytometry methodology to delineate types of cell death in airway epithelial cells. 一种适应的新型流式细胞术方法来描述气道上皮细胞的细胞死亡类型。

Journal of biological methods Pub Date : 2020-11-11 eCollection Date: 2020-01-01 DOI: 10.14440/jbm.2020.336

Samuel T Montgomery, Stephen M Stick, Anthony Kicic

{"title":"An adapted novel flow cytometry methodology to delineate types of cell death in airway epithelial cells.","authors":"Samuel T Montgomery, Stephen M Stick, Anthony Kicic","doi":"10.14440/jbm.2020.336","DOIUrl":"https://doi.org/10.14440/jbm.2020.336","url":null,"abstract":"Current methodologies to measure apoptotic and necrotic cell death using flow cytometry do not adequately differentiate between the two. Here, we describe a flow cytometry methodology adapted to airway epithelial cells (AEC) to sufficiently differentiate apoptotic and necrotic AEC. Specifically, cell lines and primary AEC (n = 12) were permeabilized or infected with rhinovirus 1b (RV1b) over 48 h. Cell death was then measured via annexin V/propidium iodide (A5/PI) or annexin V/TO-PRO-3 (A5/TP3) staining using a novel flow cytometry and gating methodology adapted to AEC. We show that A5/PI staining could not sufficiently differentiate between types of cell death following RV1b infection of primary AEC. However, A5/TP3 staining was able to distinguish six cell death populations (viable, necrotic, debris, A5+ apoptotic, A5- apoptotic, apoptotic bodies) after permeabilization or infection with RV1b, with phenotypic differences were observed in apoptotic populations. Collectively, using a staining and gating strategy never adapted to AEC, A5/TP3 could accurately differentiate and quantify viable, necrotic, and apoptotic AEC following RV1b infection.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":" ","pages":"e139"},"PeriodicalIF":0.0,"publicationDate":"2020-11-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/cd/b1/jbm-7-4-e139.PMC7666329.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38709355","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: 1

An ex vivo skin model to probe modulation of local cutaneous arachidonic acid inflammation pathway. 体外皮肤模型探讨局部皮肤花生四烯酸炎症通路的调节。

Journal of biological methods Pub Date : 2020-10-26 eCollection Date: 2020-01-01 DOI: 10.14440/jbm.2020.319

Charles M Heard

{"title":"An ex vivo skin model to probe modulation of local cutaneous arachidonic acid inflammation pathway.","authors":"Charles M Heard","doi":"10.14440/jbm.2020.319","DOIUrl":"https://doi.org/10.14440/jbm.2020.319","url":null,"abstract":"There is a need for inexpensive and reliable means to determine the modulation of cutaneous inflammation. The method outlined in this article draws together a number of scientific techniques and makes use of generally unwanted biological tissues as a means of determining skin inflammation ex vivo, and focuses on probing aspects of the arachidonic acid inflammation pathway. Freshly excised skin contains elevated levels of short-lived inducible cyclooxygenase-2 (COX-2) and, under viable conditions, COX-2 and its eicosanoid products will continue to be produced until tissue necrosis, providing a window of time in which relative levels can be probed to determine exacerbation due to an upregulating factor or downregulation due the presence of an agent exerting anti-inflammatory activity. Ex vivo porcine skin, mounted in Franz diffusion cells, is dosed topically with the xenobiotic challenge and then techniques such as Western blotting and immunohistochemistry can then be used to probe relative COX-2 levels on a semi-quantitative or qualitative level. Enzyme-linked immunosorbent assay or LCMS can be used to determine relative prostaglandin E-2 (PGE-2) levels. Thus far, the technique has been used to examine the effects of topically applied anti-inflammatories (betamethasone, ibuprofen, ketoprofen and methotrexate), natural products (fish oil, Devil's claw extract and pomegranate rind extract) and drug delivery vehicle (polyNIPAM nanogels). Topically applied xenobiotics that modulate factors such as COX-2 and PGE-2 must penetrate the intact skin, and this provides direct evidence of overcoming the \"barrier function\" of the stratum corneum in order to target the viable epidermis in sufficient levels to be able to elicit such effects. This system has particular potential as a pre-clinical screening tool for those working on the development of topical delivery systems, and has the additional advantage of being in line with 3 Rs philosophy.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":" ","pages":"e138"},"PeriodicalIF":0.0,"publicationDate":"2020-10-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/7a/54/jbm-7-4-e138.PMC7666330.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38709354","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

A simple and robust cell-based assay for the discovery of novel cytokinesis inhibitors. 一个简单和稳健的细胞为基础的分析发现新的细胞分裂抑制剂。

Journal of biological methods Pub Date : 2020-09-17 eCollection Date: 2020-01-01 DOI: 10.14440/jbm.2020.335

Laszlo Radnai, Rebecca F Stremel, Thomas Vaissiere, Li Lin, Michael Cameron, William H Martin, Gavin Rumbaugh, Theodore M Kamenecka, Patrick R Griffin, Courtney A Miller

{"title":"A simple and robust cell-based assay for the discovery of novel cytokinesis inhibitors.","authors":"Laszlo Radnai, Rebecca F Stremel, Thomas Vaissiere, Li Lin, Michael Cameron, William H Martin, Gavin Rumbaugh, Theodore M Kamenecka, Patrick R Griffin, Courtney A Miller","doi":"10.14440/jbm.2020.335","DOIUrl":"https://doi.org/10.14440/jbm.2020.335","url":null,"abstract":"Cytokinesis is the last step of mitotic cell division that separates the cytoplasm of dividing cells. Small molecule inhibitors targeting either the elements of the regulatory pathways controlling cytokinesis, or the terminal effectors have been of interest as potential drug candidates for the treatment of various diseases. Here we present a detailed protocol for a cell-based cytokinesis assay that can be used for the discovery of novel cytokinesis inhibitors. The assay is performed in a 96-well plate format in 48 h. Living cells, nuclei and nuclei of dead cells are identified by a single staining step using three fluorescent dyes, followed by rapid live cell imaging. The primary signal is the nuclei-to-cell ratio (NCR). In the presence of cytokinesis inhibitors, this ratio increases over time, as the ratio of multinucleated cells increases in the population. The ratio of dead nuclei to total nuclei provides a simultaneous measure of cytotoxicity. A screening window coefficient (Z`) of 0.65 indicates that the assay is suitable for screening purposes, as the positive and negative controls are well-separated. EC50 values can be reliably determined in a single 96-well plate by using only six different compound concentrations, enabling the testing of 4 compounds per plate. An excellent test-retest reliability (R 2 = 0.998) was found for EC50 values covering a ~1500-fold range of potencies. Established small molecule inhibitors of cytokinesis operating via direct action on actin dynamics or nonmuscle myosin II are used to demonstrate the robustness, simplicity and flexibility of the assay.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"7 3","pages":"e136"},"PeriodicalIF":0.0,"publicationDate":"2020-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/99/ad/jbm-7-3-e136.PMC7666332.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38709353","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: 1

An in vitro model of hepatic steatosis using lipid loaded induced pluripotent stem cell derived hepatocyte like cells. 利用脂质负载诱导多能干细胞衍生的肝细胞样细胞建立肝脂肪变性的体外模型。

Journal of biological methods Pub Date : 2020-07-22 eCollection Date: 2020-01-01 DOI: 10.14440/jbm.2020.330

Hiraganahalli Bhaskar Deepak, Nellikalaya Shreekrishna, Zaheerbasha Sameermahmood, Niranjan Naranapur Anand, Raghotham Hulgi, Juluri Suresh, Sonal Khare, Saravanakumar Dhakshinamoorthy

{"title":"An in vitro model of hepatic steatosis using lipid loaded induced pluripotent stem cell derived hepatocyte like cells.","authors":"Hiraganahalli Bhaskar Deepak, Nellikalaya Shreekrishna, Zaheerbasha Sameermahmood, Niranjan Naranapur Anand, Raghotham Hulgi, Juluri Suresh, Sonal Khare, Saravanakumar Dhakshinamoorthy","doi":"10.14440/jbm.2020.330","DOIUrl":"https://doi.org/10.14440/jbm.2020.330","url":null,"abstract":"Hepatic steatosis is a metabolic disease, characterized by selective and progressive accumulation of lipids in liver, leading to progressive non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and cirrhosis. The existing in vitro models of hepatic steatosis to elucidate the molecular mechanisms behind the onset of hepatic steatosis and to profile small molecule modulators uses lipid loaded primary hepatocytes, and cell lines like HepG2. The limitation of these models includes high variability between the different donor samples, reproducibility, and translatability to physiological context. An in vitro human hepatocyte derived model that mimics the pathophysiological changes seen in hepatic steatosis may provide an alternative tool for pre-clinical drug discovery research. We report the development of an in vitro experimental model of hepatic steatosis using human induced pluripotent stem cell (iPSC) derived hepatocytes like cells (HLC), loaded with lipids. Our data suggests that HLC carry some of the functional characteristics of primary hepatocytes and are amenable for development of an in vitro steatosis model using lipid loading method. The in vitro experimental model of hepatic steatosis was further characterized using biomarker analysis and validated using telmisartan. With some refinement and additional validation, our in vitro steatosis model system may be useful for profiling small molecule inhibitors and studying the mechanism of action of new drugs.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"7 3","pages":"e135"},"PeriodicalIF":0.0,"publicationDate":"2020-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/a9/be/jbm-7-3-e135.PMC7483829.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38385287","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: 2