Bio-protocol最新文献_第5页

Detection and Quantification of Calcium Ions in the Endoplasmic Reticulum and Cytoplasm of Cultured Cells Using Fluorescent Reporter Proteins and ImageJ Software. 利用荧光报告蛋白和ImageJ软件检测和定量培养细胞内质网和细胞质中的钙离子。

Bio-protocol Pub Date : 2023-08-20 DOI: 10.21769/BioProtoc.4738

Shunsuke Saito, Kazutoshi Mori

{"title":"Detection and Quantification of Calcium Ions in the Endoplasmic Reticulum and Cytoplasm of Cultured Cells Using Fluorescent Reporter Proteins and ImageJ Software.","authors":"Shunsuke Saito, Kazutoshi Mori","doi":"10.21769/BioProtoc.4738","DOIUrl":"https://doi.org/10.21769/BioProtoc.4738","url":null,"abstract":"This protocol describes a method for detecting and quantifying calcium ions in the endoplasmic reticulum (ER) and cytoplasm of cultured cells using fluorescent reporter proteins and ImageJ software. Genetically engineered fluorescent reporter proteins, such as G-CEPIA1er and GCaMP6f, localize to intracellular regions of interest (i.e., ER and cytoplasm) and emit green fluorescence upon binding to calcium ions. In this way, the fluorescence brightness of cells transfected with expression vectors for these reporters reflects the calcium ion concentration in each intracellular region. Here, we describe procedures for observing cultured cells expressing these fluorescent reporters under a fluorescence microscope, analyzing the obtained image using the free image analysis software ImageJ (https://imagej.net/ij/index.html), and determining the average fluorescence brightness of multiple cells present in the image. The current method allows us to quickly and easily quantify calcium ions on an image containing multiple cells and to determine whether there are relative differences in intracellular calcium ion concentration among experiments with different conditions. Key features Detection and quantification of calcium ions in the ER and cytoplasm using fluorescent reporter proteins Quick and easy verification of measurement results using ImageJ Simultaneous comparison between various experimental conditions (drug treatment, mutants, etc.).","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 16","pages":"e4738"},"PeriodicalIF":0.0,"publicationDate":"2023-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10450730/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10165077","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

Caste Transition and Reversion in Harpegnathos saltator Ant Colonies. Harpegnathos盐场蚂蚁殖民地的种姓转换和逆转。

Bio-protocol Pub Date : 2023-08-20 DOI: 10.21769/BioProtoc.4770

Comzit Opachaloemphan, Francisco Carmona-Aldana, Hua Yan

{"title":"Caste Transition and Reversion in Harpegnathos saltator Ant Colonies.","authors":"Comzit Opachaloemphan, Francisco Carmona-Aldana, Hua Yan","doi":"10.21769/BioProtoc.4770","DOIUrl":"10.21769/BioProtoc.4770","url":null,"abstract":"Living organisms possess the ability to respond to environmental cues and adapt their behaviors and physiologies for survival. Eusocial insects, such as ants, bees, wasps, and termites, have evolved advanced sociality: living together in colonies where individuals innately develop into reproductive and non-reproductive castes. These castes exhibit remarkably distinct behaviors and physiologies that support their specialized roles in the colony. Among ant species, Harpegnathos saltator females stand out with their highly plastic caste phenotypes that can be easily manipulated in a laboratory environment. In this protocol, we provide detailed instructions on how to generate H. saltator ant colonies, define castes based on behavioral and physiological phenotypes, and experimentally induce caste switches, including the transition from a non-reproductive worker to a reproductive gamergate and vice versa (known as reversion). The unusual features of H. saltator make it a valuable tool to investigate cellular and molecular mechanisms underlying phenotypic plasticity in eusocial organisms. Key features H. saltator is one of few ant species showing remarkable caste plasticity with striking phenotypic changes, being a useful subject for studying behavioral plasticity. Caste switches in H. saltator can be easily manipulated in a controlled laboratory environment by controlling the presence of reproductive females in a colony. The relatively large size of H. saltator females allows researchers to dissect various tissues of interest and conduct detailed phenotypic analyses.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 16","pages":"e4770"},"PeriodicalIF":0.0,"publicationDate":"2023-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/84/ef/BioProtoc-13-16-4770.PMC10450750.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10465677","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

T Cell Clonal Analysis Using Single-cell RNA Sequencing and Reference Maps. 利用单细胞RNA测序和参考图谱进行T细胞克隆分析。

Bio-protocol Pub Date : 2023-08-20 DOI: 10.21769/BioProtoc.4735

Massimo Andreatta, Paul Gueguen, Nicholas Borcherding, Santiago J Carmona

{"title":"T Cell Clonal Analysis Using Single-cell RNA Sequencing and Reference Maps.","authors":"Massimo Andreatta, Paul Gueguen, Nicholas Borcherding, Santiago J Carmona","doi":"10.21769/BioProtoc.4735","DOIUrl":"https://doi.org/10.21769/BioProtoc.4735","url":null,"abstract":"T cells are endowed with T-cell antigen receptors (TCR) that give them the capacity to recognize specific antigens and mount antigen-specific adaptive immune responses. Because TCR sequences are distinct in each naïve T cell, they serve as molecular barcodes to track T cells with clonal relatedness and shared antigen specificity through proliferation, differentiation, and migration. Single-cell RNA sequencing provides coupled information of TCR sequence and transcriptional state in individual cells, enabling T-cell clonotype-specific analyses. In this protocol, we outline a computational workflow to perform T-cell states and clonal analysis from scRNA-seq data based on the R packages Seurat, ProjecTILs, and scRepertoire. Given a scRNA-seq T-cell dataset with TCR sequence information, cell states are automatically annotated by reference projection using the ProjecTILs method. TCR information is used to track individual clonotypes, assess their clonal expansion, proliferation rates, bias towards specific differentiation states, and the clonal overlap between T-cell subtypes. We provide fully reproducible R code to conduct these analyses and generate useful visualizations that can be adapted for the needs of the protocol user. Key features Computational analysis of paired scRNA-seq and scTCR-seq data Characterizing T-cell functional state by reference-based analysis using ProjecTILs Exploring T-cell clonal structure using scRepertoire Linking T-cell clonality to transcriptomic state to study relationships between clonal expansion and functional phenotype Graphical overview.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 16","pages":"e4735"},"PeriodicalIF":0.0,"publicationDate":"2023-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10450729/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10483840","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

A Method for Studying Social Signal Learning of the Waggle Dance in Honey Bees. 蜜蜂摇摆舞社会信号学习的研究方法。

Bio-protocol Pub Date : 2023-08-20 DOI: 10.21769/BioProtoc.4789

Shihao Dong, Tao Lin, James C Nieh, Ken Tan

{"title":"A Method for Studying Social Signal Learning of the Waggle Dance in Honey Bees.","authors":"Shihao Dong, Tao Lin, James C Nieh, Ken Tan","doi":"10.21769/BioProtoc.4789","DOIUrl":"https://doi.org/10.21769/BioProtoc.4789","url":null,"abstract":"Honey bees use a complex form of spatial referential communication. Their waggle dance communicates to nestmates the direction, distance, and quality of a resource by encoding celestial cues, retinal optic flow, and relative food value into motion and sound within the nest. This protocol was developed to investigate the potential for social learning of this waggle dance. Using this protocol, we showed that correct waggle dancing requires social learning. Bees (Apis mellifera) that did not follow any dances before they first danced produced significantly more disordered dances, with larger waggle angle divergence errors, and encoded distance incorrectly. The former deficits improved with experience, but distance encoding was set for life. The first dances of bees that could follow other dancers had none of these impairments. Social learning, therefore, shapes honey bee signaling, as it does communication in human infants, birds, and multiple other vertebrate species. However, much remains to be learned about insects' social learning, and this protocol will help to address knowledge gaps in the understanding of sophisticated social signal learning, particularly in understanding the molecular bases for such learning. Key features It was unclear if honey bees (Apis mellifera) could improve their waggle dance by following experienced dancers before they first waggle dance. Honey bees perform their first waggle dances with more errors if they cannot follow experienced waggle dancers first. Directional and disorder errors improved over time, but distance error was maintained. Bees in experimental colonies continued to communicate longer distances than control bees. Dancing correctly, with less directional error and disorder, requires social learning. Distance encoding in the honey bee dance is largely genetic but may also include a component of cultural transmission.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 16","pages":"e4789"},"PeriodicalIF":0.0,"publicationDate":"2023-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/4b/13/BioProtoc-13-16-4789.PMC10450786.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10465679","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

Quantification of Botrytis cinerea Growth in Arabidopsis thaliana. 拟南芥灰霉病菌生长的定量研究。

Bio-protocol Pub Date : 2023-08-20 DOI: 10.21769/BioProtoc.4740

Patricia Scholz, Kent D Chapman, Till Ischebeck, Athanas Guzha

{"title":"Quantification of Botrytis cinerea Growth in Arabidopsis thaliana.","authors":"Patricia Scholz, Kent D Chapman, Till Ischebeck, Athanas Guzha","doi":"10.21769/BioProtoc.4740","DOIUrl":"https://doi.org/10.21769/BioProtoc.4740","url":null,"abstract":"Yield losses attributed to plant pathogens pose a serious threat to plant productivity and food security. Botrytis cinerea is one of the most devastating plant pathogens, infecting a wide array of plant species; it has also been established as a model organism to study plant-pathogen interactions. In this context, development of different assays to follow the relative success of B. cinerea infections is required. Here, we describe two methods to quantify B. cinerea development in Arabidopsis thaliana genotypes through measurements of lesion development and quantification of fungal genomic DNA in infected tissues. This provides two independent techniques that are useful in assessing the susceptibility or tolerance of different Arabidopsis genotypes to B. cinerea. Key features Protocol for the propagation of the necrotrophic plant pathogen fungus Botrytis cinerea and spore production. Two methods of Arabidopsis thaliana infection with the pathogen using droplet and spray inoculation. Two readouts, either by measuring lesion size or by the quantification of fungal DNA using quantitative PCR. The two methods are applicable across plant species susceptible the B. cinerea. Graphical overview A simplified overview of the droplet and spray infection methods used for the determination of B. cinerea growth in different Arabidopsis genotypes.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 16","pages":"e4740"},"PeriodicalIF":0.0,"publicationDate":"2023-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/61/db/BioProtoc-13-16-4740.PMC10450733.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10465672","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

Analysis of Pectin-derived Monosaccharides from Arabidopsis Using GC-MS. 气相色谱-质谱法分析拟南芥果胶单糖。

Bio-protocol Pub Date : 2023-08-20 DOI: 10.21769/BioProtoc.4746

Patricia Scholz, Kent D Chapman, Till Ischebeck, Athanas Guzha

{"title":"Analysis of Pectin-derived Monosaccharides from Arabidopsis Using GC-MS.","authors":"Patricia Scholz, Kent D Chapman, Till Ischebeck, Athanas Guzha","doi":"10.21769/BioProtoc.4746","DOIUrl":"https://doi.org/10.21769/BioProtoc.4746","url":null,"abstract":"Pectin is a complex polysaccharide present in the plant cell wall, whose composition is constantly remodelled to adapt to environmental or developmental changes. Mutants with altered pectin composition have been reported to exhibit altered stress or pathogen resistance. Understanding the link between mutant phenotypes and their pectin composition requires robust analytical methods to detect changes in the relative monosaccharide composition. Here, we describe a quick and efficient gas chromatography-mass spectrometry (GC-MS)-based method that allows the differential analysis of pectin monosaccharide composition in plants under different conditions or between mutant plants and their respective wild types. Pectin is extracted from seed mucilage or from the alcohol-insoluble residue prepared from leaves or other organs and is subsequently hydrolysed with trifluoracetic acid. The resulting acidic and neutral monosaccharides are then derivatised and measured simultaneously by GC-MS. Key features Comparative analysis of monosaccharide content in Arabidopsis-derived pectin between different genotypes or different treatments. Procedures for two sources of pectin are shown: seed coat mucilage and alcohol-insoluble residue. Allows quick analyses of neutral and acidic monosaccharides simultaneously. Graphical overview.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 16","pages":"e4746"},"PeriodicalIF":0.0,"publicationDate":"2023-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10450732/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10465673","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

Stereotactic Delivery of Helper-dependent Adenoviral Viral Vectors at Distinct Developmental Time Points to Perform Age-dependent Molecular Manipulations of the Mouse Calyx of Held. 在不同的发育时间点立体定向递送辅助依赖性腺病毒载体以对Hold的小鼠Calyx进行年龄依赖性分子操作。

Bio-protocol Pub Date : 2023-08-20 DOI: 10.21769/BioProtoc.4793

Christian Keine, Mohammed Al-Yaari, Tamara Radulovic, Samuel M Young

{"title":"Stereotactic Delivery of Helper-dependent Adenoviral Viral Vectors at Distinct Developmental Time Points to Perform Age-dependent Molecular Manipulations of the Mouse Calyx of Held.","authors":"Christian Keine, Mohammed Al-Yaari, Tamara Radulovic, Samuel M Young","doi":"10.21769/BioProtoc.4793","DOIUrl":"10.21769/BioProtoc.4793","url":null,"abstract":"Synapses are specialized structures that enable neuronal communication, which is essential for brain function and development. Alterations in synaptic proteins have been linked to various neurological and neuropsychiatric disorders. Therefore, manipulating synaptic proteins in vivo can provide insight into the molecular mechanisms underlying these disorders and aid in developing new therapeutic strategies. Previous methods such as constitutive knock-out animals are limited by developmental compensation and off-target effects. The current approach outlines procedures for age-dependent molecular manipulations in mice using helper-dependent adenovirus viral vectors (HdAd) at distinct developmental time points. Using stereotactic injection of HdAds in both newborn and juvenile mice, we demonstrate the versatility of this method to express Cre recombinase in globular bushy cells of juvenile Rac1fl/fl mice to ablate presynaptic Rac1 and study its role in synaptic transmission. Separately, we overexpress CaV2 α1 subunits at two distinct developmental time points to elucidate the mechanisms that determine presynaptic CaV2 channel abundance and preference. This method presents a reliable, cost-effective, and minimally invasive approach for controlling gene expression in specific regions of the mouse brain and will be a powerful tool to decipher brain function in health and disease. Key features Virus-mediated genetic perturbation in neonatal and young adult mice. Stereotaxic injection allows targeting of brain structures at different developmental stages to study the impact of genetic perturbation throughout the development.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 16","pages":"e4793"},"PeriodicalIF":0.0,"publicationDate":"2023-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/69/79/BioProtoc-13-16-4793.PMC10450731.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10483844","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

Perforated Patch Clamp Recordings in ex vivo Brain Slices from Adult Mice. 成年小鼠离体脑切片穿孔膜片钳记录。

Bio-protocol Pub Date : 2023-08-20 DOI: 10.21769/BioProtoc.4741

Simon Hess, Helmut Wratil, Peter Kloppenburg

引用次数: 0

A Simple Sonication Method to Isolate the Chloroplast Lumen in Arabidopsis thaliana. 拟南芥叶绿体管腔的简单超声分离方法研究。

Bio-protocol Pub Date : 2023-08-05 DOI: 10.21769/BioProtoc.4756

Jingfang Hao, Alizée Malnoë

{"title":"A Simple Sonication Method to Isolate the Chloroplast Lumen in Arabidopsis thaliana.","authors":"Jingfang Hao, Alizée Malnoë","doi":"10.21769/BioProtoc.4756","DOIUrl":"https://doi.org/10.21769/BioProtoc.4756","url":null,"abstract":"The chloroplast lumen contains at least 80 proteins whose function and regulation are not yet fully understood. Isolating the chloroplast lumen enables the characterization of the lumenal proteins. The lumen can be isolated in several ways through thylakoid disruption using a Yeda press or sonication, or through thylakoid solubilization using a detergent. Here, we present a simple procedure to isolate thylakoid lumen by sonication using leaves of the plant Arabidopsis thaliana. The step-by-step procedure is as follows: thylakoids are isolated from chloroplasts, loosely associated thylakoid surface proteins from the stroma are removed, and the lumen fraction is collected in the supernatant following sonication and centrifugation. Compared to other procedures, this method is easy to implement and saves time, plant material, and cost. Lumenal proteins are obtained in high quantity and purity; however, some stromal membrane-associated proteins are released to the lumen fraction, so this method could be further adapted if needed by decreasing sonication power and/or time.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 15","pages":"e4756"},"PeriodicalIF":0.0,"publicationDate":"2023-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/07/31/BioProtoc-13-15-4756.PMC10415170.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9998478","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

Development of a Mouse Model of Hematopoietic Loss of Y Chromosome. Y染色体造血缺失小鼠模型的建立。

Bio-protocol Pub Date : 2023-08-05 DOI: 10.21769/BioProtoc.4729

Soichi Sano, Kenneth Walsh

引用次数: 0