Bio-protocol最新文献

{"title":"High-resolution Cryo-EM Structure Determination of a-Synuclein-A Prototypical Amyloid Fibril.","authors":"Juan C Sanchez, Joshua A Pierson, Collin G Borcik, Chad M Rienstra, Elizabeth R Wright","doi":"10.21769/BioProtoc.5171","DOIUrl":"10.21769/BioProtoc.5171","url":null,"abstract":"<p><p>The physiological role of a-synuclein (a-syn), an intrinsically disordered presynaptic neuronal protein, is believed to impact the release of neurotransmitters through interactions with the SNARE complex. However, under certain cellular conditions that are not well understood, a-syn will self-assemble into β-sheet-rich fibrils that accumulate and form insoluble neuronal inclusions. Studies of patient-derived brain tissues have concluded that these inclusions are associated with Parkinson's disease, the second most common neurodegenerative disorder, and other synuclein-related diseases called synucleinopathies. In addition, repetitions of specific mutations to the SNCA gene, the gene that encodes a-syn, result in an increased disposition for synucleinopathies. The latest advances in cryo-EM structure determination and real-space helical reconstruction methods have resulted in over 60 in vitro structures of a-syn fibrils solved to date, with a handful of these reaching a resolution below 2.5 Å. Here, we provide a protocol for a-syn protein expression, purification, and fibrilization. We detail how sample quality is assessed by negative stain transmission electron microscopy (NS-TEM) analysis and followed by sample vitrification using the Vitrobot Mark IV vitrification robot. We provide a detailed step-by-step protocol for high-resolution cryo-EM structure determination of a-syn fibrils using RELION and a series of specialized helical reconstruction tools that can be run within RELION. Finally, we detail how ChimeraX, Coot, and Phenix are used to build and refine a molecular model into the high-resolution cryo-EM map. This workflow resulted in a 2.04 Å structure of a-syn fibrils with excellent resolution of residues 36-97 and an additional island of density for residues 15-22 that had not been previously reported. This workflow should serve as a starting point for individuals new to the neurodegeneration and structural biology fields. Together, this procedure lays the foundation for advanced structural studies of a-syn and other amyloid fibrils. Key features • In vitro fibril amplification method yielding twisting fibrils that span several micrometers in length and are suitable for cryo-EM structure determination. • High-throughput cryo-EM data collection of neurodegenerative fibrils, such as alpha-synuclein. • Use of RELION implementations of helical reconstruction algorithms to generate high-resolution 3D structures of a-synuclein fibrils. • Brief demonstration of the use of ChimeraX, Coot, and Phenix for molecular model building and refinement.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5171"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825309/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434528","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}

{"title":"Using the <i>Sleeping Beauty</i> Transposon System for Doxycycline-inducible Gene Expression in RAW264.7 Macrophage Cells to Study Phagocytosis.","authors":"Parsa Kamali, Gregory D Fairn","doi":"10.21769/BioProtoc.5178","DOIUrl":"10.21769/BioProtoc.5178","url":null,"abstract":"<p><p>Macrophages are known for engulfing and digesting pathogens and dead cells through a specialized form of endocytosis called phagocytosis. Unfortunately, many macrophage cell lines are refractory to most reagents used for transient transfections. Alternative transient approaches, such as electroporation or transduction with lentiviral vectors, typically cause cell death (electroporation) or can be time-consuming to generate numerous lentivirus when using different genes of interest. Therefore, we use the <i>Sleeping Beauty</i> system to generate stably transfected cells. The system uses a \"resurrected\" transposase gene named <i>Sleeping Beauty</i> found in salmonid fish. Experimentally, the system introduces two plasmids: one carrying the <i>Sleeping Beauty</i> transposase and the other with an integration cassette carrying the gene of interest, a reverse-doxycycline controlled repressor gene, and an antibiotic resistance gene. The construct used in this protocol provides puromycin resistance. Stable integrations are selected by culturing the cells in the presence of puromycin, and further enrichment can be obtained using fluorescence-activated cell sorting (FACS). In this protocol, we use the <i>Sleeping Beauty</i> transposon system to generate RAW264.7 cells with doxycycline-inducible inositol polyphosphate 4-phosphatase B containing a C-terminal CaaX motif (INPP4B-CaaX). INPP4B-CaaX dephosphorylates the D-4 position of phosphatidylinositol 3,4-<i>bis</i>phosphate and inhibits phagocytosis. One benefit is that generating stable cell lines is substantially faster than selecting for random integrations. Without FACS, the method typically gives ~50% of the cells that are transfected; with sorting, this approaches 100%. This makes phagocytosis experiments easier since more cells can be analyzed per experiment, allowing for population-based measurements where a ~10% transient transfection rate is insufficient. Finally, using the doxycycline-promoter allows for low near endogenous expression of proteins or robust overexpression. Key features • This protocol builds on the protocols and reagents developed by Kowarz et al. [1] and extends it to using RAW macrophages. • Allows for the rapid generation of stably induced cell lines. • This protocol also determines the phagocytic index and efficiency.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5178"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825295/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434667","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}

{"title":"Versatile Click Chemistry-based Approaches to Illuminate DNA and RNA G-Quadruplexes in Human Cells.","authors":"Angélique Pipier, David Monchaud","doi":"10.21769/BioProtoc.5209","DOIUrl":"10.21769/BioProtoc.5209","url":null,"abstract":"<p><p>The existence and functional relevance of DNA and RNA G-quadruplexes (G4s) in human cells is now beyond debate, but how did we reach such a level of confidence? Thanks to a panoply of molecular tools and techniques that are now routinely implemented in wet labs. Among them, G4 imaging ranks high because of its reliability and practical convenience, which now makes cellular G4 detection quick and easy; also, because this technique is sensitive and responsive to any G4 modulations in cells, which thus allows gaining precious insights into G4 biology. Herein, we briefly explain what a G4 is and how they can be visualized in human cells; then, we present the strategy we have been developing for several years now for in situ click G4 imaging, which relies on the use of biomimetic G4 ligands referred to as TASQs (for template-assembled synthetic G-quartets) and is far more straightforward and modular than classically used immunodetection methods. We thus show why and how to illuminate G4s with TASQs and provide a detailed, step-by-step methodology (including the preparation of the materials, the methodology per se, and a series of notes to address any possible pitfalls that may arise during the experiments) to make G4 imaging ever easier to operate. Key features • MultiTASQs are clickable probes usable for the detection of cellular DNA and RNA G-quadruplex (G4). • In situ click chemistry relies on the labeling of G4 by clickable probes once in their cellular binding sites. • Experiments can be performed by incubating the clickable probe either in live cells or in fixed cells. • The published but unoptimized protocol is now totally revised to allow for reliable G4 detection in human cells.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5209"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825307/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434686","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}

{"title":"Model Architecture Analysis and Implementation of TENET for Cell-Cell Interaction Network Reconstruction Using Spatial Transcriptomics Data.","authors":"Ziyang Wang, Yujian Lee, Yongqi Xu, Peng Gao, Chuckel Yu, Jiaxing Chen","doi":"10.21769/BioProtoc.5205","DOIUrl":"10.21769/BioProtoc.5205","url":null,"abstract":"<p><p>Cellular communication relies on the intricate interplay of signaling molecules, which come together to form the cell-cell interaction (CCI) network that orchestrates tissue behavior. Researchers have shown that shallow neural networks can effectively reconstruct the CCI from the abundant molecular data captured in spatial transcriptomics (ST). However, in scenarios characterized by sparse connections and excessive noise within the CCI, shallow networks are often susceptible to inaccuracies, leading to suboptimal reconstruction outcomes. To achieve a more comprehensive and precise CCI reconstruction, we propose a novel method called triple-enhancement-based graph neural network (TENET). The TENET framework has been implemented and evaluated on both real and synthetic ST datasets. This protocol primarily introduces our network architecture and its implementation. Key features • Cell-cell reconstruction network using ST data. • To facilitate the implementation of a more holistic CCI, we incorporate diverse CCI modalities into consideration. • To further enrich the input information, the downstream gene regulatory network (GRN) is also incorporated as an input to the network. • The network architecture considers global and local cellular and genetic features rather than solely leveraging the graph neural network (GNN) to model such information.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5205"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11833462/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143451339","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}

{"title":"Protocol to Identify Unknown Flanking DNA Using Partially Overlapping Primer-based PCR for Genome Walking.","authors":"Mengya Jia, Dongqin Ding, Xiaohua Liu, Haixing Li","doi":"10.21769/BioProtoc.5172","DOIUrl":"10.21769/BioProtoc.5172","url":null,"abstract":"<p><p>Genome walking is a popular molecular technique for accessing unknown flanking DNAs, which has been widely used in biology-related fields. Herein, a simple but accurate genome-walking protocol named partially overlapping primer (POP)-based PCR (POP-PCR) is described. This protocol exploits a POP set of three POPs to mediate genome walking. The three POPs have a 10 nt 3' overlap and 15 nt heterologous 5' regions. Therefore, a POP can partially anneal to the previous POP site only at a relatively low temperature (approximately 50 °C). In primary POP-PCR, the low-temperature (25 °C) cycle allows the primary POP to partially anneal to site(s) of an unknown flank and many sites of the genome, synthesizing many single-stranded DNAs. In the subsequent high-temperature (65 °C) cycle, the target single-stranded DNA is converted into double-stranded DNA by the sequence-specific primer, attributed to the presence of this primer complement, while non-target single-stranded DNA cannot become double-stranded because it lacks a binding site for both primers. As a result, only the target DNA is amplified in the remaining 65 °C cycles. In secondary or tertiary POP-PCR, the 50 °C cycle directs the POP to the previous POP site and synthesizes many single-stranded DNAs. However, as in the primary PCR, only the target DNA can be amplified in the subsequent 65 °C cycles. This POP-PCR protocol has many potential applications, such as screening microbes, identifying transgenic sites, or mining new genetic resources. Key features • This POP-PCR protocol, built upon the technique developed by Li et al. [1], is universal to genome walking of any species. • The established protocol relies on the 10 nt 3' overlap among a set of three POPs. • The first two rounds of POP-PCRs can generally give a positive walking outcome.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5172"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825308/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434565","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}

{"title":"Detection of Amylin-β-amyloid Hetero-Oligomers by Enzyme-Linked Immunosorbent Assay.","authors":"Noah S Leibold, Deepak Kotiya, Nirmal Verma, Florin Despa","doi":"10.21769/BioProtoc.5179","DOIUrl":"10.21769/BioProtoc.5179","url":null,"abstract":"<p><p>Amylin is an amyloidogenic neuroendocrine hormone co-synthesized and co-secreted with insulin from the pancreas. It readily crosses the blood-brain barrier and synergistically forms mixed amyloid plaques with β-amyloid (Aβ) in brain parenchyma. Parenchymal amylin-Aβ plaques are found in both sporadic and early-onset familial Alzheimer's disease (AD), yet their (patho)physiological role remains elusive, particularly due to a lack of detection modalities for these mixed plaques. Previously, we developed an enzyme-linked immunosorbent assay (ELISA) capable of detecting amylin-Aβ hetero-oligomers in brain lysate and blood using a polyclonal anti-amylin antibody to capture hetero-oligomers and a monoclonal anti-Aβ mid-domain detection antibody combination. This combination allows for the recognition of distinct amylin epitopes, which remain accessible after amylin-Aβ oligomerization has begun, and precise detection of Aβ epitopes available after oligomer formation. The utility of this assay is evidenced in our previous report, wherein differences in hetero-oligomer content in brain tissue from patients with and without AD and patients with and without diabetes were distinguished. Additionally, using AD model rats, we provided evidence that our assay can be employed for the detection of amylin-Aβ in blood. This assay and protocol are important innovations in the field of AD research because they meet an unmet need to detect mixed amyloid plaques that, if targeted therapeutically, could reduce AD progression and severity. Key features • Detects amylin-Aβ hetero-oligomers in blood from patients with Alzheimer's disease. • Enables simultaneous, high-throughput analysis of hetero-oligomer content of brain and blood tissue. • Allows exploration into the amylin-Aβ interaction during AD pathogenesis, potentially leading to novel treatment mechanisms by controlling the amylin-Aβ interaction.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5179"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825304/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434599","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}

{"title":"Flood Inoculation of <i>Fusarium eumartii</i> in Tomato Seedlings: Method for Evaluating the Infectivity of Pathogen Spores.","authors":"María Cecilia Terrile, Florencia Anabel Mesas, María Elisa Picco, María Florencia Salcedo, Andrea Yamila Mansilla","doi":"10.21769/BioProtoc.5198","DOIUrl":"10.21769/BioProtoc.5198","url":null,"abstract":"<p><p>The <i>Fusarium</i> genus includes various fungi of great significance in agriculture. <i>Fusarium solani</i> f. sp. <i>eumartii (F. eumartii</i>), traditionally known as a potato pathogen, has also been identified as a cause of disease in tomatoes. This protocol provides a detailed, efficient, and robust flood-inoculation method for assessing <i>F. eumartii</i> infection of young tomato seedlings grown on MS medium plates. It includes the evaluation of the lesion area and the quantification of the remaining fungal inoculum in tomato seedlings. In summary, the straightforwardness and efficiency of this bioassay make it a powerful quantitative tool for selecting fungicidal compounds or defense response inducers in tomato plants, offering a promising approach with significant potential for preventing fungal diseases in crops. Key features • Accelerated fungal infection method in tomato seedlings, shortening overall experimental time. • Allows simultaneous evaluation of fungal infectivity and quantitative remaining inoculum. • Easily adaptable for screening fungicides and defense inducers in various plant-pathogen interactions.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5198"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825292/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434527","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}

{"title":"Identification of <i>Mycobacterium tuberculosis</i> and its Drug Resistance by Targeted Nanopore Sequencing Technology.","authors":"Chen Tang, Feng Xu, Xiaoqun Zheng, Guangxin Xiang","doi":"10.21769/BioProtoc.5170","DOIUrl":"10.21769/BioProtoc.5170","url":null,"abstract":"<p><p>Tuberculosis (TB) remains the leading cause of human mortality in infectious diseases. Drug-resistant TB, particularly multidrug-resistant TB and extensively drug-resistant TB, poses a pressing clinical and public health challenge. The main causative agents of TB are known as <i>Mycobacterium tuberculosis</i> (MTB), which exhibits a highly complex drug resistance profile. Traditional culture-based phenotypic drug susceptibility testing is time-consuming, and PCR-based assays are restricted to detecting known mutational hotspots. In this study, we present a protocol leveraging high-throughput nanopore sequencing technology in conjunction with multiplex PCR, termed targeted nanopore sequencing, for the identification of MTB and analysis of its drug resistance. Our method for MTB drug resistance assessment offers the benefits of being culture-free, efficient, high-throughput, and highly accurate, which could significantly aid in clinical patient management and the control of TB infections. Key features • Targeted nanopore sequencing detects 18 genes simultaneously linked to antibiotic resistance in MTB. • The method provides broad drug resistance profiles for 14 first- and second-line anti-TB drugs without bacterial culture. • The expedited turnaround time of the process is approximately 7.5 h with a detection limit of 10² bacteria/mL.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5170"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825305/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434529","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}

{"title":"Profiling the Secretome of Glioblastoma Cells Under Histone Deacetylase Inhibition Using Mass Spectrometry.","authors":"Aline Menezes, Yara Martins, Fábio César Sousa Nogueira, Denise de Abreu Pereira, Katia Carneiro","doi":"10.21769/BioProtoc.5197","DOIUrl":"10.21769/BioProtoc.5197","url":null,"abstract":"<p><p>Glioblastoma (GBM) is the most aggressive brain tumor, and different efforts have been employed in the search for new drugs and therapeutic protocols for GBM. A label-free, mass spectrometry-based quantitative proteomics has been developed to identify and characterize proteins that are differentially expressed in GBM to gain a better understanding of the interactions and functions that lead to the pathological state focusing on the extracellular matrix (ECM). The main challenge in GBM research has been to identify novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. To better investigate the GBM secretome upon in vitro treatment with histone deacetylase inhibitor (iHDAC), we employed a high-throughput label-free methodology of protein identification and quantification based on mass spectrometry followed by in silico studies. Our analysis revealed significant changes in the ECM protein profile, particularly those associated with the angiogenic matrisome. Proteins such as decorin, ADAM10, ADAM12, and ADAM15 were differentially regulated upon in silico analysis. In contrast, key angiogenesis markers such as VEGF and ECM proteins like fibronectin and integrins did not display significant changes. These results suggest that iHDAC inhibitors may modulate or suppress tumor behavior growth by targeting ECM proteins' secretion rather than directly inhibiting angiogenesis. Key features • Analysis of the secretome of U87MG glioblastoma cells. • Studies of mass spectrometry designed to modulate GBM biology and behavior focused on histone deacetylase inhibitors (iHDAC). • Mass spectrometry was developed to identify and characterize proteins that are differentially expressed in GBM.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5197"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825294/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434476","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}

{"title":"FlashTag-mediated Labeling for Intraventricular Macrophages in the Embryonic Brain.","authors":"Hisa Asai, Mizuki Ono, Takaki Miyata, Yuki Hattori","doi":"10.21769/BioProtoc.5166","DOIUrl":"10.21769/BioProtoc.5166","url":null,"abstract":"<p><p>The fate mapping technique is essential for understanding how cells differentiate and organize into complex structures. Various methods are used in fate mapping, including dye injections, genetic labeling (e.g., Cre-lox recombination systems), and molecular markers to label cells and track their progeny. One such method, the FlashTag system, was originally developed to label neural progenitors. This technique involves injecting carboxyfluorescein diacetate succinimidyl ester (CFSE) into the lateral ventricles of mouse embryos, relying on the direct uptake of dye by cells. The injection of CFSE into the lateral ventricle allows for the pulse labeling of mitotic (M-phase) neural progenitors in the ventricular zone and their progeny throughout the brain. This approach enables us to trace the future locations and differentiation paths of neural progenitors. In our previous study, we adapted this method to selectively label central nervous system-associated macrophages (CAMs) in the lateral ventricle by using a lower concentration of CFSE compared to the original protocol. Microglia, the brain's immune cells, which play pivotal roles in both physiological and pathological contexts, begin colonizing the brain around embryonic day (E) 9.5 in mice, with their population expanding as development progresses. The modified FlashTag technique allowed us to trace the fate of intraventricular CAMs, revealing that certain populations of microglia are derived from these cells. The optimized approach offers deeper insights into the developmental trajectories of microglia. This protocol outlines the modified FlashTag method for labeling intraventricular CAMs, detailing the CFSE injection procedure, evaluation of CFSE dilution, and preparation of tissue for immunohistochemistry. Key features • This protocol builds upon the method developed by Govindan et al. and extends its application to intraventricular CAMs. • This protocol allows for the cell fate tracking of intraventricular CAMs within 24 h. • This protocol requires the technique of intraventricular injection of CFSE into embryonic brains.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 2","pages":"e5166"},"PeriodicalIF":1.0,"publicationDate":"2025-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11769748/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143054557","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}