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Protocol for the isolation of human neutrophil-derived extracellular vesicles for characterization and functional experimentation.
IF 1.3
STAR Protocols Pub Date : 2025-04-04 DOI: 10.1016/j.xpro.2025.103739
Robert Richter, Ezgi Sari, Delores Stacks, Crystal Lewis, Julian Smith, Ningyong Xu, Tamiajoi Langster, Dakota Finley, Camilla Margaroli, Kristopher R Genschmer
{"title":"Protocol for the isolation of human neutrophil-derived extracellular vesicles for characterization and functional experimentation.","authors":"Robert Richter, Ezgi Sari, Delores Stacks, Crystal Lewis, Julian Smith, Ningyong Xu, Tamiajoi Langster, Dakota Finley, Camilla Margaroli, Kristopher R Genschmer","doi":"10.1016/j.xpro.2025.103739","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103739","url":null,"abstract":"<p><p>Small extracellular vesicles (EVs) derived from neutrophils play an important role in the remodeling of extracellular matrices in human health and disease. Here, we present a protocol for isolating EVs secreted from human neutrophils either ex vivo or within circulation. We describe steps for neutrophil and plasma isolation, neutrophil activation, and EV isolation and enumeration. We then detail procedures for isolating CD66b<sup>+</sup> EVs using magnetic bead pull-down and subsequent characterization by flow cytometry, western blot, and neutrophil elastase activity assays. For complete information on the generation and use of this protocol, please refer to Genschmer et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103739"},"PeriodicalIF":1.3,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143789194","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol to determine the topology of integral endoplasmic reticulum membrane protein in Saccharomyces cerevisiae.
IF 1.3
STAR Protocols Pub Date : 2025-04-03 DOI: 10.1016/j.xpro.2025.103727
Ritika Chaudhary, Vineet Choudhary
{"title":"Protocol to determine the topology of integral endoplasmic reticulum membrane protein in Saccharomyces cerevisiae.","authors":"Ritika Chaudhary, Vineet Choudhary","doi":"10.1016/j.xpro.2025.103727","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103727","url":null,"abstract":"<p><p>Here, we present a protocol to determine the topology of Fld1 (few lipid droplets), an integral endoplasmic reticulum (ER) membrane protein in Saccharomyces cerevisiae. We describe steps to generate functional N-terminal GFP and C-terminal mCherry fusion with Fld1. We detail the strategy to perform subcellular fractionation to isolate ER-derived microsomes that were subjected to salt detergent extraction analysis. We then provide procedures to determine the topology of Fld1 using proteinase K treatment.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103727"},"PeriodicalIF":1.3,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143789272","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for the establishment of a mouse myocardial infarction and ischemia-reperfusion model via heart compression.
IF 1.3
STAR Protocols Pub Date : 2025-04-03 DOI: 10.1016/j.xpro.2025.103724
Chongning Zhong, Hui Dong, Xianwu Cheng, Hailian Quan, Lan Hong
{"title":"Protocol for the establishment of a mouse myocardial infarction and ischemia-reperfusion model via heart compression.","authors":"Chongning Zhong, Hui Dong, Xianwu Cheng, Hailian Quan, Lan Hong","doi":"10.1016/j.xpro.2025.103724","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103724","url":null,"abstract":"<p><p>Myocardial infarction (MI) and myocardial ischemia-reperfusion injury (MIRI) are major pathological conditions in cardiovascular disease, requiring in-depth study for effective therapy development. Here, we present a detailed protocol for establishing a mouse model using the squeeze technique to simulate MI and MIRI. Key steps include isoflurane-induced anesthesia, left anterior descending artery (LAD) ligation, and real-time monitoring. Additionally, we describe procedures for histological analysis, offering a comprehensive approach to investigating disease mechanisms and potential therapeutic strategies. For complete details on the use and execution of this protocol, please refer to Gao et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103724"},"PeriodicalIF":1.3,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143784523","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for observing tunneling nanotube formation and function in both fixed and live primary mouse neurons and microglia coculture system.
IF 1.3
STAR Protocols Pub Date : 2025-04-03 DOI: 10.1016/j.xpro.2025.103723
Vivian Baker, Dimitri Budinger, Sean-Patrick Riechers, Michael T Heneka
{"title":"Protocol for observing tunneling nanotube formation and function in both fixed and live primary mouse neurons and microglia coculture system.","authors":"Vivian Baker, Dimitri Budinger, Sean-Patrick Riechers, Michael T Heneka","doi":"10.1016/j.xpro.2025.103723","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103723","url":null,"abstract":"<p><p>Microglia and neurons can connect via tunneling nanotubes (TNTs), facilitating the transfer of organelles, vesicles, and proteins. Here, we present a protocol for visualizing murine TNT formation and material transfer between neurons and microglia in both fixed samples and samples for live-cell imaging, as well as for flow cytometry. We describe steps for identifying and measuring TNTs and quantifying the transport of aggregated proteins, such as α-synuclein or tau, between these cells. For complete details on the use and execution of this protocol, please refer to Scheiblich et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103723"},"PeriodicalIF":1.3,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143784519","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for identification of proteins from deyolked zebrafish embryos.
IF 1.3
STAR Protocols Pub Date : 2025-04-03 DOI: 10.1016/j.xpro.2025.103728
Kathiresan Purushothaman, Saraphina Dianne Tneo Rwei Qing, Yong Xin Ee, Nithiyakala Ananthakrishnan, Shanmugam Harshiktha, Shubha Vij, Qingsong Lin, Igor Babiak
{"title":"Protocol for identification of proteins from deyolked zebrafish embryos.","authors":"Kathiresan Purushothaman, Saraphina Dianne Tneo Rwei Qing, Yong Xin Ee, Nithiyakala Ananthakrishnan, Shanmugam Harshiktha, Shubha Vij, Qingsong Lin, Igor Babiak","doi":"10.1016/j.xpro.2025.103728","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103728","url":null,"abstract":"<p><p>Zebrafish is a key model for studying vertebrate development and human diseases, but proteomic data during embryogenesis are limited due to interference from egg yolk proteins. Here, we present a protocol for the isolation, identification, and analysis of proteins from zebrafish embryos. We describe steps for dechorionation, deyolking, protein extraction, SDS-PAGE, and trypsin digestion. We then detail procedures for peptide separation using liquid chromatography-tandem mass spectrometry (LC-MS/MS), identification via ProteinPilot, and functional analysis with Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology. For complete details on the use and execution of this protocol, please refer to Purushothaman et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103728"},"PeriodicalIF":1.3,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143784517","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for label-free quantitative lysine lactylproteome profiling.
IF 1.3
STAR Protocols Pub Date : 2025-04-03 DOI: 10.1016/j.xpro.2025.103726
Yu Ran, Qiqing Yang, Lianqi Zhou, Heyu Li, Bing Yang, Long Zhang
{"title":"Protocol for label-free quantitative lysine lactylproteome profiling.","authors":"Yu Ran, Qiqing Yang, Lianqi Zhou, Heyu Li, Bing Yang, Long Zhang","doi":"10.1016/j.xpro.2025.103726","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103726","url":null,"abstract":"<p><p>L-lactate has been recognized as an essential molecule for signaling and metabolic balance. Lactylation, a post-translational modification (PTM) derived from L-lactate, is commonly observed on various proteins and plays essential roles in cellular processes. Here, we present a protocol to globally profile the lactylation proteome and perform label-free quantification. We provide steps for cell preparation, protein extraction, digestion, peptide desalting, and lactylpeptide enrichment. Additionally, we outline the parameters for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. For complete details on the use and execution of this protocol, please refer to Li et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103726"},"PeriodicalIF":1.3,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143784518","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for renal subcapsular islet transplantation in diabetic mice to induce long-term immune tolerance.
IF 1.3
STAR Protocols Pub Date : 2025-04-03 DOI: 10.1016/j.xpro.2025.103729
Yinglin Yuan, Qibin Wu, Guoli Huai, Shengyun Ma, Gaoping Zhao
{"title":"Protocol for renal subcapsular islet transplantation in diabetic mice to induce long-term immune tolerance.","authors":"Yinglin Yuan, Qibin Wu, Guoli Huai, Shengyun Ma, Gaoping Zhao","doi":"10.1016/j.xpro.2025.103729","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103729","url":null,"abstract":"<p><p>Murine renal subcapsular islet transplantation presents a promising technique for diabetes treatment by addressing challenges such as immune rejection and reliance on immunosuppressive drugs. Here, we present a protocol for the isolation, purification, and transplantation of mouse pancreatic islets that overcomes these challenges. Specifically, we describe steps for inducing diabetes with streptozotocin, pancreatic perfusion and isolation, and islet cell purification. We then detail procedures for renal subcapsular islet transplantation, dual antibody therapy, and immune cell and graft monitoring. For complete details on the use and execution of this protocol, please refer to Liu et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103729"},"PeriodicalIF":1.3,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143789193","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Amplex red assay, a standardized in vitro protocol to quantify the efficacy of autotaxin inhibitors.
IF 1.3
STAR Protocols Pub Date : 2025-04-01 DOI: 10.1016/j.xpro.2025.103721
Elli-Anna Stylianaki, Christiana Magkrioti, Eleanna Kaffe, Alexios N Matralis, Vassilis Aidinis
{"title":"Amplex red assay, a standardized in vitro protocol to quantify the efficacy of autotaxin inhibitors.","authors":"Elli-Anna Stylianaki, Christiana Magkrioti, Eleanna Kaffe, Alexios N Matralis, Vassilis Aidinis","doi":"10.1016/j.xpro.2025.103721","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103721","url":null,"abstract":"<p><p>Autotaxin (ATX), a secreted lysophospholipase D responsible for the extracellular production of the bioactive phospholipid lysophosphatidic acid (LPA), is a therapeutic target in idiopathic pulmonary fibrosis and pancreatic cancer, among other disorders, promoting the synthesis of novel ATX inhibitors. Here, we present a protocol for detecting and characterizing ATX inhibitors using a fluorometry-based microplate assay. We describe steps for a first screening of compounds, half-maximal inhibitory concentration (IC<sub>50</sub>) quantification of initial hits, screening for false positives, and identification of the hits' mode of inhibition. For complete details on the use and execution of this protocol, please refer to Stylianaki et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103721"},"PeriodicalIF":1.3,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143781542","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for detecting nutrient uptake and photosynthate partitioning in rice seedling using a non-invasive system. 利用非侵入式系统检测水稻幼苗养分吸收和光合作用分配的规程。
IF 1.3
STAR Protocols Pub Date : 2025-04-01 DOI: 10.1016/j.xpro.2025.103707
Jinzhi Li, He Zhang, Jianming Gao
{"title":"Protocol for detecting nutrient uptake and photosynthate partitioning in rice seedling using a non-invasive system.","authors":"Jinzhi Li, He Zhang, Jianming Gao","doi":"10.1016/j.xpro.2025.103707","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103707","url":null,"abstract":"<p><p>A non-invasive system can measure nutrient uptake and photosynthate partitioning simultaneously while allowing organisms to metabolize without interference in the process of measurement. Here, we present a protocol for detecting nutrient uptake and photosynthate partitioning in rice seedling using a non-invasive system. We describe steps for preparing plant material and material for the non-invasive system. We then detail procedures for setting up and applying the non-invasive measurement system. For complete details on the use and execution of this protocol, please refer to Li et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103707"},"PeriodicalIF":1.3,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143774259","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human hematopoietic stem and progenitor cells.
IF 1.3
STAR Protocols Pub Date : 2025-03-31 DOI: 10.1016/j.xpro.2025.103722
Lucrezia Della Volpe, Roberta Vacca, Raffaella Di Micco
{"title":"Protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human hematopoietic stem and progenitor cells.","authors":"Lucrezia Della Volpe, Roberta Vacca, Raffaella Di Micco","doi":"10.1016/j.xpro.2025.103722","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103722","url":null,"abstract":"<p><p>Long-range correction strategies require ex vivo activation of hematopoietic stem and progenitor cells (HSPCs) to engage the homology-directed repair (HDR) mechanism, but prolonged culture causes harmful cellular responses, reducing the long-term functionality of gene-edited (GE) HSPCs. Here, we present a protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human HSPCs. We describe steps for HSPC thawing, ex vivo treatments, gene editing, and downstream in vitro and in vivo analyses to assess the functionality of GE-HSPCs. For complete details on the use and execution of this protocol, please refer to della Volpe et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103722"},"PeriodicalIF":1.3,"publicationDate":"2025-03-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143774268","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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