Bio-protocol最新文献_第4页

Preparation of Human Kidney Progenitor Cultures and Their Differentiation into Podocytes. 人肾祖细胞培养物的制备及其向足细胞的分化。

Bio-protocol Pub Date : 2023-08-20 DOI: 10.21769/BioProtoc.4757

Maria Elena Melica, Maria Lucia Angelotti, Giulia Antonelli, Anna J Peired, Carolina Conte, Letizia De Chiara, Benedetta Mazzinghi, Elena Lazzeri, Laura Lasagni, Paola Romagnani

引用次数: 0

Determination of Poly(3-hydroxybutyrate) Content in Cyanobacterium Synechocystis sp. PCC 6803 Using Acid Hydrolysis Followed by High-performance Liquid Chromatography. 酸水解-高效液相色谱法测定聚藻藻PCC 6803中聚(3-羟基丁酸酯)含量

Bio-protocol Pub Date : 2023-08-20 DOI: 10.21769/BioProtoc.4790

Janine Kaewbai-Ngam, Aran Incharoensakdi, Tanakarn Monshupanee

引用次数: 0

Catheterization of Pulmonary and Carotid Arteries for Concurrent Measurement of Mean Pulmonary and Systemic Arterial Pressure in Rat Models of Pulmonary Arterial Hypertension. 在肺动脉高压大鼠模型中导管插入肺动脉和颈动脉以同时测量平均肺动脉压和全身动脉压

Bio-protocol Pub Date : 2023-08-20 DOI: 10.21769/BioProtoc.4737

Tanoy Sarkar, Ayman Isbatan, Sakib M Moinuddin, Jiwang Chen, Fakhrul Ahsan

{"title":"Catheterization of Pulmonary and Carotid Arteries for Concurrent Measurement of Mean Pulmonary and Systemic Arterial Pressure in Rat Models of Pulmonary Arterial Hypertension.","authors":"Tanoy Sarkar, Ayman Isbatan, Sakib M Moinuddin, Jiwang Chen, Fakhrul Ahsan","doi":"10.21769/BioProtoc.4737","DOIUrl":"10.21769/BioProtoc.4737","url":null,"abstract":"Pulmonary hypertension (PH) is a group of pulmonary vascular disorders in which mean pulmonary arterial pressure (mPAP) becomes abnormally high because of various pathological conditions, including remodeling of the pulmonary arteries, lung and heart disorders, or congenital conditions. Various animal models, including mouse and rat models, have been used to recapitulate elevated mPAP observed in PH patients. However, the measurement and recording of mPAP and mean systemic arterial pressure (mSAP) in small animals require microsurgical procedures and a sophisticated data acquisition system. In this paper, we describe the surgical procedures for right heart catheterizations (RHC) to measure mPAP in rats. We also explain the catheterization of the carotid artery for simultaneous measurement of mPAP and mSAP using the PowerLab Data Acquisition system. We enumerate the surgical steps involved in exposing the jugular vein and the carotid artery for catheterizing these two blood vessels. We list the tools used for microsurgery in rats, describe the methods for preparing catheters, and illustrate the process for inserting the catheters in the pulmonary and carotid arteries. Finally, we delineate the steps involved in the calibration and setup of the PowerLab system for recording both mPAP and mSAP. This is the first protocol wherein we meticulously explain the surgical procedures for RHC in rats and the recording of mPAP and mSAP. We believe this protocol will be essential for PH research. Investigators with little training in animal handling can reproduce this microsurgical procedure for RHC in rats and measure mPAP and mSAP in rat models of PH. Further, this protocol is likely to help master RHC in rats that are performed for other conditions, such as heart failure, congenital heart disease, heart valve disorders, and heart transplantation.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 16","pages":"e4737"},"PeriodicalIF":0.0,"publicationDate":"2023-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10461069/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10118311","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

Mass Spectrometry-based Lipidomics, Lipid Bioenergetics, and Web Tool for Lipid Profiling and Quantification in Human Cells. 质谱为基础的脂质组学，脂质生物能量学，和网络工具脂质分析和定量在人类细胞。

Bio-protocol Pub Date : 2023-08-20 DOI: 10.21769/BioProtoc.4742

Liang Cui, Meisam Yousefi, Xin Yap, Clara W T Koh, Kwan Sing Leona Tay, Yaw Shin Ooi, Kuan Rong Chan

{"title":"Mass Spectrometry-based Lipidomics, Lipid Bioenergetics, and Web Tool for Lipid Profiling and Quantification in Human Cells.","authors":"Liang Cui, Meisam Yousefi, Xin Yap, Clara W T Koh, Kwan Sing Leona Tay, Yaw Shin Ooi, Kuan Rong Chan","doi":"10.21769/BioProtoc.4742","DOIUrl":"https://doi.org/10.21769/BioProtoc.4742","url":null,"abstract":"Lipids can play diverse roles in metabolism, signaling, transport across membranes, regulating body temperature, and inflammation. Some viruses have evolved to exploit lipids in human cells to promote viral entry, fusion, replication, assembly, and energy production through fatty acid beta-oxidation. Hence, studying the virus-lipid interactions provides an opportunity to understand the biological processes involved in the viral life cycle, which can facilitate the development of antivirals. Due to the diversity and complexity of lipids, the assessment of lipid utilization in infected host cells can be challenging. However, the development of mass spectrometry, bioenergetics profiling, and bioinformatics has significantly advanced our knowledge on the study of lipidomics. Herein, we describe the detailed methods for lipid extraction, mass spectrometry, and assessment of fatty acid oxidation on cellular bioenergetics, as well as the bioinformatics approaches for detailed lipid analysis and utilization in host cells. These methods were employed for the investigation of lipid alterations in TMEM41B- and VMP1-deficient cells, where we previously found global dysregulations of the lipidome in these cells. Furthermore, we developed a web app to plot clustermaps or heatmaps for mass spectrometry data that is open source and can be hosted locally or at https://kuanrongchan-lipid-metabolite-analysis-app-k4im47.streamlit.app/. This protocol provides an efficient step-by-step methodology to assess lipid composition and usage in host cells.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 16","pages":"e4742"},"PeriodicalIF":0.0,"publicationDate":"2023-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10450793/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10483841","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 Semi-throughput Procedure for Assaying Plant NADP-malate Dehydrogenase Activity Using a Plate Reader. 利用平板阅读器测定植物nadp -苹果酸脱氢酶活性的半通量方法。

Bio-protocol Pub Date : 2023-08-20 DOI: 10.21769/BioProtoc.4769

Kevin Baudry, Emmanuelle Issakidis-Bourguet

引用次数: 0

Quantification of Chromosomal Aberrations in Mammalian Cells. 哺乳动物细胞中染色体畸变的定量分析。

Bio-protocol Pub Date : 2023-08-20 DOI: 10.21769/BioProtoc.4739

Inés Paniagua, Jacqueline J L Jacobs

{"title":"Quantification of Chromosomal Aberrations in Mammalian Cells.","authors":"Inés Paniagua, Jacqueline J L Jacobs","doi":"10.21769/BioProtoc.4739","DOIUrl":"https://doi.org/10.21769/BioProtoc.4739","url":null,"abstract":"Maintenance of genome integrity requires efficient and faithful resolution of DNA breaks and DNA replication obstacles. Dysfunctions in any of the processes orchestrating such resolution can lead to chromosomal instability, which appears as numerical and structural chromosome aberrations. Conventional cytogenetics remains as the golden standard method to detect naturally occurring chromosomal aberrations or those resulting from the treatment with genotoxic drugs. However, the success of cytogenetic studies depends on having high-quality chromosome spreads, which has been proven to be particularly challenging. Moreover, a lack of scoring guidelines and standardized methods for treating cells with genotoxic agents contribute to significant variability amongst different studies. Here, we report a simple and effective method for obtaining well-spread chromosomes from mammalian cells for the analysis of chromosomal aberrations. In this method, cells are (1) arrested in metaphase (when chromosome morphology is clearest), (2) swollen in hypotonic solution, (3) fixed before being dropped onto microscope slides, and (4) stained with DNA dyes to visualize the chromosomes. Metaphase chromosomes are then analyzed using high-resolution microscopy. We also provide examples, representative images, and useful guidelines to facilitate the scoring of the different chromosomal aberrations. This method can be used for the diagnosis of genetic diseases, as well as for cancer studies, by identifying chromosomal defects and providing insight into the cellular processes that influence chromosome integrity.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 16","pages":"e4739"},"PeriodicalIF":0.0,"publicationDate":"2023-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10450737/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10109817","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

Multi-color Flow Cytometry Protocol to Characterize Myeloid Cells in Mouse Retina Research. 小鼠视网膜髓系细胞的多色流式细胞术研究。

Bio-protocol Pub Date : 2023-08-20 DOI: 10.21769/BioProtoc.4745

Wei Xiao, Rami A Shahror, Carol A Morris, Ruth B Caldwell, Abdelrahman Y Fouda

{"title":"Multi-color Flow Cytometry Protocol to Characterize Myeloid Cells in Mouse Retina Research.","authors":"Wei Xiao, Rami A Shahror, Carol A Morris, Ruth B Caldwell, Abdelrahman Y Fouda","doi":"10.21769/BioProtoc.4745","DOIUrl":"10.21769/BioProtoc.4745","url":null,"abstract":"Myeloid cells, specifically microglia and macrophages, are activated in retinal diseases and can improve or worsen retinopathy outcomes based on their inflammatory phenotype. However, assessing the myeloid cell response after retinal injury in mice remains challenging due to the small tissue size and the challenges of distinguishing microglia from infiltrating macrophages. In this protocol paper, we describe a flow cytometry-based protocol to assess retinal microglia/macrophage and their inflammatory phenotype after injury. The protocol is amenable to the incorporation of other markers of interest to other researchers. Key features This protocol describes a flow cytometry-based method to analyze the myeloid cell response in retinopathy mouse models. The protocol can distinguish between microglia- and monocyte-derived macrophages. It can be modified to incorporate markers of interest. We show representative results from three different retinopathy models, namely ischemia-reperfusion injury, endotoxin-induced uveitis, and oxygen-induced retinopathy.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 16","pages":"e4745"},"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/50/b9/BioProtoc-13-16-4745.PMC10450788.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10109818","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

Fluorescent Biosensor Imaging of Nitrate in Arabidopsis thaliana. 拟南芥硝酸盐的荧光生物传感器成像。

Bio-protocol Pub Date : 2023-08-20 DOI: 10.21769/BioProtoc.4743

Yen-Ning Chen, Cheng-Hsun Ho

{"title":"Fluorescent Biosensor Imaging of Nitrate in Arabidopsis thaliana.","authors":"Yen-Ning Chen, Cheng-Hsun Ho","doi":"10.21769/BioProtoc.4743","DOIUrl":"https://doi.org/10.21769/BioProtoc.4743","url":null,"abstract":"Nitrate (NO3-) is an essential element and nutrient for plants and animals. Despite extensive studies on the regulation of nitrate uptake and downstream responses in various cells, our knowledge of the distribution of nitrogen forms in different root cell types and their cellular compartments is still limited. Previous physiological models have relied on in vitro biochemistry and metabolite level analysis, which limits the ability to differentiate between cell types and compartments. Here, to address this, we report a nuclear-localized, genetically encoded fluorescent biosensor, which we named nlsNitraMeter3.0, for the quantitative visualization of nitrate concentration and distribution at the cellular level in Arabidopsis thaliana. This biosensor was specifically designed for nitrate measurements, not nitrite. Through genetic engineering to create and select sensors using yeast, Xenopus oocyte, and Arabidopsis expression systems, we developed a reversible and highly specific nitrate sensor. This method, combined with fluorescence imaging systems such as confocal microscopy, allows for the understanding and monitoring of nitrate transporter activity in plant root cells in a minimally invasive manner. Furthermore, this approach enables the functional analysis of nitrate transporters and the measurement of nitrate distribution in plants, providing a valuable tool for plant biology research. In summary, we provide a protocol for sensor development and a biosensor that can be used to monitor nitrate levels in plants. Key features This protocol builds upon the concept of FRET biosensors for in vivo visualization of spatiotemporal nitrate levels at a cellular resolution. Nitrate levels can be quantified utilizing the biosensor in conjunction with either a plate reader or a fluorescence microscope.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 16","pages":"e4743"},"PeriodicalIF":0.0,"publicationDate":"2023-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10450734/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10109819","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

In vitro Analysis of Stalled Ribosomes Using Puromycin Incorporation. 利用嘌呤霉素整合技术体外分析停滞的核糖体

Bio-protocol Pub Date : 2023-08-20 DOI: 10.21769/BioProtoc.4744

MaKenzie R Scarpitti, Michael G Kearse

{"title":"In vitro Analysis of Stalled Ribosomes Using Puromycin Incorporation.","authors":"MaKenzie R Scarpitti, Michael G Kearse","doi":"10.21769/BioProtoc.4744","DOIUrl":"10.21769/BioProtoc.4744","url":null,"abstract":"Ribosome footprint profiling has demonstrated that ribosomes can be slowed or stalled on select mRNAs, often due to the presence of rare codons, stalling motifs, or via a ribosome-binding protein (e.g., FMRP). Stalled ribosomes can act as physical roadblocks for trailing ribosomes and ultimately can cause ribosome collisions that stimulate no-go mRNA decay. Detecting stalled or slowed ribosomes in cells by ribosome footprint profiling or classic polysome profiling is laborious, technically challenging, and low throughput. Here, we present a protocol to assay for stalled ribosomes on in vitro-transcribed reporter mRNAs using a robust, commercially available mammalian in vitro translation lysate and an optimized low-speed sucrose cushion. In short, we take advantage of the ability of puromycin to incorporate into the nascent polypeptide and cause the ribosome to dissociate from the mRNA during active elongation, as well as the ability to selectively pellet ribosomes through a low-speed sucrose cushion due to their large molecular weight. Stalled ribosomes are not actively elongating and do not incorporate puromycin, allowing the ribosome-bound mRNA to pellet in the low-speed sucrose cushion. RT-qPCR is used to quantify the amount of ribosome-bound reporter mRNA in the pellet. This workflow allows for direct assessment of stalled ribosomes and is fully amendable to insertion of putative stalling motifs in the target mRNA, as well as supplementation with recombinant proteins or small molecule inhibitors that target translation elongation. Key features This protocol is optimized for cap-dependent in vitro translation in the dynamic linear range. Details for generating capped reporter mRNA in one day are provided. Requires as little as one day to complete if starting with in vitro-transcribed mRNA. This protocol requires access to an ultracentrifuge and a real-time PCR system.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 16","pages":"e4744"},"PeriodicalIF":0.0,"publicationDate":"2023-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10450738/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10109821","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

Improved Methods for Acetocarmine and Haematoxylin Staining to Visualize Chromosomes in the Filamentous Green Alga Zygnema (Charophyta). 改进乙酰卡明和血苏木精染色方法，以观察丝状绿藻(Charophyta)中的染色体。

Bio-protocol Pub Date : 2023-08-20 DOI: 10.21769/BioProtoc.4768

Nina Rittmeier, Andreas Holzinger

{"title":"Improved Methods for Acetocarmine and Haematoxylin Staining to Visualize Chromosomes in the Filamentous Green Alga Zygnema (Charophyta).","authors":"Nina Rittmeier, Andreas Holzinger","doi":"10.21769/BioProtoc.4768","DOIUrl":"10.21769/BioProtoc.4768","url":null,"abstract":"Genome sizes of Zygnema spp. vary greatly, being unknown whether polyploidization occurred. The exact number of chromosomes in this genus is unknown since counting methods established for higher plants cannot be applied to green algae. The massive presence of pectins and arabinogalactan proteins in the cell wall interferes with the uptake of staining solutions; moreover, cell divisions in green algae are not restricted to meristems as in higher plants, which is another limiting factor. Cell divisions occur randomly in the thallus, due to the intercalary growth of algal filaments. Therefore, we increased the number of cell divisions via synchronization by changing the light cycle (10:14 h light/dark). The number of observed mitotic stages peaked at the beginning of the dark cycle. This protocol describes two methods for the visualization of chromosomes in the filamentous green alga Zygnema. Existing protocols were modified, leading to improved acetocarmine and haematoxylin staining methods as investigated by light microscopy. A freeze-shattering approach with liquid nitrogen was applied to increase the accessibility of the haematoxylin dye. These modified protocols allowed reliable chromosome counting in the genus Zygnema. Key features Improved method for chromosome staining in filamentous green algae. Optimized for the Zygnema strains SAG 698-1a (Z. cylindricum), SAG 698-1b (Z. circumcarinatum), and SAG 2419 (Zygnema 'Saalach'). This protocol builds upon the methods of chromosomal staining in green algae developed by Wittmann (1965), Staker (1971), and Fujii and Guerra (1998). Cultivation and synchronization: 14 days; fixation and permeabilization: 24 h; staining: 1 h; image analysis and chromosome number quantification: up to 20 h.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 16","pages":"e4768"},"PeriodicalIF":0.0,"publicationDate":"2023-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10450781/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10483837","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