Bio-protocol最新文献

{"title":"Primary Mouse Choroidal Endothelial Cell Culture.","authors":"Qiuhua Yang, Yongfeng Cai, Qian Ma, Yuqing Huo","doi":"10.21769/BioProtoc.5355","DOIUrl":"10.21769/BioProtoc.5355","url":null,"abstract":"<p><p>The study of choroidal endothelial cells is essential for understanding the pathological mechanisms underlying choroidal neovascularization and other vision-threatening disorders. Traditional methods for isolating and culturing primary endothelial cells often yield mixed populations or require specialized equipment, limiting their widespread use. Here, we present a straightforward protocol for isolating and culturing primary mouse choroidal endothelial cells. This protocol involves enzymatic digestion of choroidal tissue, magnetic-activated cell sorting (MACS) to enrich CD31⁺ endothelial cells, and optimized culture conditions to promote cell proliferation and maintain endothelial phenotype. The protocol is strategic, reproducible, and requires minimal specialized equipment, making it accessible for researchers across various fields. By providing a robust method for obtaining pure choroidal endothelial cell cultures, this protocol facilitates the study of cell-specific behaviors and responses, advancing research into choroidal vascular diseases. Key features • Describes a protocol for isolating mouse choroidal endothelial cells (mCECs) using Matrigel^TM-based explant culture followed by CD31⁺ cell enrichment. • Utilizes enzymatic dissociation with dispase and filtration to achieve a single-cell suspension, ensuring high cell yield and purity. • Confirms endothelial cell identity, enabling reliable downstream applications. • Supports experimental induction of endothelial-to-mesenchymal transition (EndMT) using mouse transforming growth factor β2 (TGFβ2), making it suitable for studying vascular remodeling processes.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 12","pages":"e5355"},"PeriodicalIF":1.0,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12222637/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144577231","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":"Surgical Implantation of a Telemetry-Based Pressure Sensor in the Internal Jugular Vein to Monitor Respiration Wirelessly.","authors":"Neha Kushwaha, Debanjan Dasgupta","doi":"10.21769/BioProtoc.5343","DOIUrl":"10.21769/BioProtoc.5343","url":null,"abstract":"<p><p>Active sampling, such as respiration, is known to play a major role in modulating how sensory information is perceived and encoded in the field of olfaction. Hence, monitoring respiration is crucial for understanding olfactory-guided behavior and physiology. Several methods used to measure respiration, such as infrared cameras, piezoelectric sensors, video monitoring, temperature probes, intubation, and intranasal cannula, require the animal or at least its head to be fixed. However, telemetry-based sensors can be used wirelessly, allowing animals to move freely. Here, we describe the surgical protocol to implant a telemetry pressure sensor in the internal jugular vein to detect changes in thoracic pressure. The sensor can thus help in monitoring respiration by transmitting the signal wirelessly. We describe a way of inserting the probe into the right jugular vein aseptically while housing the transmitter underneath the skin on the back of the animal. Next, based on the optimal spot for the best signal, we secure the position of the probe and suture the skin. The animal then undergoes regular post-operative care with painkillers and soft diets for up to a week. The method offers two main advantages; first, it uses a strategy similar to the jugular vein catheterization, which is widely established in rodents. Second, it minimizes the need for extensive post-operative care, including not having to shift to a liquid diet post-recovery. This makes the animals fit for most behavioral experiments requiring water or food restrictions. Key features • Based on Dasgupta et al. [1], this protocol describes the surgical procedure involved in the aseptic implantation of the telemetry-based pressure sensor in the internal jugular vein. • Reduces the need for extensive post-operative care and eliminates dietary restrictions post-recovery. • The procedure helps in obtaining mice with an implanted sensor for wireless respiration recording.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 12","pages":"e5343"},"PeriodicalIF":1.0,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12254592/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144628081","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":"Method for Extracellular Electrochemical Impedance Spectroscopy on Epithelial Cell Monolayers.","authors":"Athena J Chien, Colby F Lewallen, Hanna Khor, Analia Vazquez Cegla, Rongming Guo, Adrienne L Watson, Chris Hatcher, Nael A McCarty, Kapil Bharti, Craig R Forest","doi":"10.21769/BioProtoc.5341","DOIUrl":"10.21769/BioProtoc.5341","url":null,"abstract":"<p><p>Epithelial tissues form barriers to the flow of ions, nutrients, waste products, bacteria, and viruses. The conventional electrophysiology measurement of transepithelial resistance (TEER/TER) can quantify epithelial barrier integrity, but does not capture all the electrical behavior of the tissue or provide insight into membrane-specific properties. Electrochemical impedance spectroscopy, in addition to measurement of TER, enables measurement of transepithelial capacitance (TEC) and a ratio of electrical time constants for the tissue, which we term the membrane ratio. This protocol describes how to perform galvanostatic electrochemical impedance spectroscopy on epithelia using commercially available cell culture inserts and chambers, detailing the apparatus, electrical signal, fitting technique, and error quantification. The measurement can be performed in under 1 min on commercially available cell culture inserts and electrophysiology chambers using instrumentation capable of galvanostatic sinusoidal signal processing (4 μA amplitude, 2 Hz to 50 kHz). All fits to the model have less than 10 Ω mean absolute error, revealing repeatable values distinct for each cell type. On representative retinal pigment (n = 3) and bronchiolar epithelial samples (n = 4), TER measurements were 500-667 Ω·cm² and 955-1,034 Ω·cm² (within the expected range), TEC measurements were 3.65-4.10 μF/cm² and 1.07-1.10 μF/cm², and membrane ratio measurements were 18-22 and 1.9-2.2, respectively. Key features • This protocol requires preexisting experience with culturing epithelial cells (such as Caco-2, RPE, and 16HBE) for a successful outcome. • Builds upon methods by Lewallen et al. [1] and Linz et al. [2], integrating commercial chambers and providing a quantitative estimate of error. • Provides code to run measurement, process data, and report error; requires access to MATLAB software, but no coding experience is necessary. • Allows for repeated measurements on the same sample. Graphical overview <b>Electrochemical impedance spectroscopy measurement involves sending a galvanostatic signal through the electrophysiology chamber and across the epithelial cell monolayer (left) and results in complex impedance data at each frequency.</b> This data is then fit to an electrical circuit model to output transepithelial resistance (TER), transepithelial capacitance (TEC), and membrane ratio (α) (right).</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 12","pages":"e5341"},"PeriodicalIF":1.0,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12254589/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144628078","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":"Ub-POD: A Ubiquitin-Specific Proximity-Dependent Labeling Technique to Identify E3 Ubiquitin Ligase Substrates in Human Cells.","authors":"Urbi Mukhopadhyay, Sophie Levantovsky, Christian Behrends, Sagar Bhogaraju","doi":"10.21769/BioProtoc.5351","DOIUrl":"10.21769/BioProtoc.5351","url":null,"abstract":"<p><p>Ubiquitination is a post-translational protein modification that regulates a vast majority of processes during protein homeostasis. The covalent attachment of ubiquitin is a highly regulated process carried out by the sequential action of the three enzymes E1, E2, and E3. E3 ligases share a dual function of 1) transferring covalently attached ubiquitin from the catalytic cysteine of E2 (E2~Ub) to the substrate and 2) providing substrate specificity. Our current knowledge of their individual substrate pools is incomplete due to the difficult capture of these transient substrate-E3 ligase interactions. Here, we present an efficient protocol that enables the selective biotinylation of substrates of a given ubiquitin ligase. In brief, the candidate E3 ligase is fused to the biotin ligase BirA and ubiquitin to a biotin acceptor peptide, an Avi-tag variant (-2) AP. Cells are co-transfected with these fusion constructs and exposed to biotin, resulting in a BirA-E3 ligase-catalyzed biotinylation of (-2) AP-Ub when in complex with E2. As the next step, the biotinylated (-2) AP-Ub is transferred covalently to the substrate lysine, which enables an enrichment via denaturing streptavidin pulldown. Substrate candidates can then be identified via mass spectrometry (MS). Our ubiquitin-specific proximity-dependent labeling (Ub-POD) method allows robust biotinylation of the ubiquitylation substrates of a candidate E3 ligase thanks to the wild-type BirA and biotin acceptor peptide fused to the E3 and Ub, respectively. Because of the highly Ub-specific labeling, Ub-POD is more appropriate for identifying ubiquitination substrates compared to other conventional proximity labeling or immunoprecipitation (IP) approaches. Key features • A simple and cost-effective method using common chemicals makes Ub-POD easy to implement in any laboratory. • Can be an exploratory tool to identify new substrates via mass spectrometry (MS) or as a validation tool in combination with immunoblotting or immunofluorescence. • Knowledge of triggers and constraints of E3 ligase activity is beneficial.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 12","pages":"e5351"},"PeriodicalIF":1.0,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12222633/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144577234","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":"An Optimized Ex Vivo Protocol for Quantitative Electrophysiological Assessment of Neuromuscular Junctions and Skeletal Muscle Function Using the Aurora System.","authors":"Can Cui, Zhengyuan Bao, Simon Kwoon-Ho Chow, Qianjin Wang, Senlin Chai, Zhihong Xu, Qing Jiang, Wing Hoi Cheung","doi":"10.21769/BioProtoc.5353","DOIUrl":"10.21769/BioProtoc.5353","url":null,"abstract":"<p><p>The neuromuscular junction (NMJ) is critical for muscle function, and its dysfunction underlies conditions such as sarcopenia and motor neuron diseases. Current protocols for assessing NMJ function often lack standardized stimulation parameters, limiting reproducibility. This study presents an optimized ex vivo method to evaluate skeletal muscle and NMJ function using the Aurora Scientific system, incorporating validated stimulation protocols for both nerve and muscle to ensure consistency. Key steps include tissue preparation in a low-calcium, high-magnesium solution to preserve NMJ integrity, determination of optimal muscle length, and sequential stimulation protocols to quantify neurotransmission failure and intratetanic fatigue. By integrating rigorous standardization, this approach enhances reproducibility and precision, providing a robust framework for investigating NMJ pathophysiology in aging and disease models. Key features • Dual stimulation modes enable direct muscle and indirect nerve stimulation to isolate NMJ-specific dysfunction. • Optimized stimulation parameters for nerve (5 mA, 0.8 ms pulse width) and muscle (300 mA, 0.2 ms pulse width) on mouse model. • Preservation of NMJ integrity through dissection in low-calcium, high-magnesium artificial cerebrospinal fluid (aCSF) and synthetic interstitial fluid (SIF). • Quantitative analysis of neurotransmission failure and intratetanic fatigue using standardized equations. <b>This protocol is used in:</b> Ageing Cell (2024), DOI: 10.1111/acel.14156.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 12","pages":"e5353"},"PeriodicalIF":1.0,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12229795/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144585778","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":"Cryopreservation of Bulk-Produced Primary Rat Oligodendrocyte Progenitor Cells.","authors":"Hanki Kim, Ramsha Afzal, Bum Jun Kim, Hyo Jin Cho, Jun Young Choi","doi":"10.21769/BioProtoc.5345","DOIUrl":"10.21769/BioProtoc.5345","url":null,"abstract":"<p><p>Primary oligodendrocyte cultures are a crucial driving force for in vitro research on oligodendrocytes (OLs) and myelin. Various methods are available to obtain oligodendrocyte lineage cells, primarily from neonatal rodent brains or human induced pluripotent stem cells (iPSCs). In this protocol, we describe a step-by-step procedure for detaching and cryopreserving primary rat oligodendrocyte progenitor cells (OPCs), followed by the thawing, proliferation, and differentiation of the cryopreserved OPCs. After freezing in a serum-free cryopreservation medium, the OPCs can be preserved at -80 °C for up to two months without notable changes in viability, proliferation, or differentiation into mature OLs. Cryopreserved OPCs can be differentiated into mature OLs with robust myelin processes and the capacity to wrap around neuron-mimicking structures. Combined with the author's method for primary OL culture, which allows for bulk production of OPCs, OPC cryopreservation may substantially improve the efficiency of in vitro OL research. Key features • This protocol recommends the use of a specific culture method that enables the simple, bulk production of primary rat OPCs. • Through this protocol, researchers may obtain large numbers of cryopreserved OPCs, which can be reserved for up to two months. • This protocol facilitates the planning of in vitro experiments and reduces the effort required to maintain adequate numbers of primary OPCs for large-scale experiments.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 12","pages":"e5345"},"PeriodicalIF":1.0,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12254587/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144628076","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":"Expression and Purification of the Human Voltage-Gated Proton Channel (hH_v1).","authors":"Emerson M Carmona, D Marien Cortes, Luis G Cuello","doi":"10.21769/BioProtoc.5344","DOIUrl":"10.21769/BioProtoc.5344","url":null,"abstract":"<p><p>The voltage-gated proton channel (H_v1) is a membrane protein that dissipates acute cell proton accumulations. To understand the molecular mechanisms explaining H_v1 function, methods for purifying the protein are needed. Previously, methods were developed for expressing and purifying human H_v1 (hH_v1) in yeast and later in bacteria. However, these methodologies produced low protein yields and had high production costs, considerably limiting their usefulness. The protocol described in this work was developed to overcome those limitations. hH_v1 is overexpressed in bacteria, solubilized with the detergent Anzergent 3-12, and purified by immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography (SEC). Our protocol produced higher protein yields at lower costs than previously published methodologies. Key features • hH_v1, containing a poly-His tag followed by an enterokinase cutting site in its N-terminus, is overexpressed in <i>E. coli</i> by autoinduction. • The detergent Anzergent 3-12 is used to solubilize and purify hH_v1 using nickel-immobilized metal affinity chromatography (IMAC). • The entire procedure can be performed in 6 days.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 12","pages":"e5344"},"PeriodicalIF":1.0,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12254590/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144628077","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":"Preparation of Testicular Cells for Immunofluorescence Analysis of Manchette in Elongating Spermatids.","authors":"Changmin Niu, Opeyemi Dhikhirullahi, Zhibing Zhang","doi":"10.21769/BioProtoc.5340","DOIUrl":"10.21769/BioProtoc.5340","url":null,"abstract":"<p><p>Immunofluorescence staining is a technique that permits the visualization of components of various cell preparations. Manchette, a transient structure that is only present in elongating spermatids, is involved in intra-manchette transport (IMT) for sperm flagella formation. Sperm flagella are assembled by intra-flagellar transport (IFT). Due to the big complexes formed by IMT and IFT components, it has been challenging to visualize these components in tissue sections. This is because the proteins that make up these complexes overlap with each other. Testicular tissue is digested by a combination of DNase I and Collagenase IV enzymes and fixed by paraformaldehyde and sucrose. After permeabilization with Triton X-100, testicular cells are incubated with specific antibodies to detect the components in the manchette and developing sperm tails. This method allows for cell type-specific resolution without interference from surrounding cells like Sertoli, Leydig, or peritubular myoid cells. Additionally, isolated cells produce cleaner immunofluorescence signals compared to other methods like tissue section/whole mount, making this method the best fit for visualizing protein localization in germ cells when spatial context is not being considered. Hence, this protocol provides the detailed methodology for isolating male mice germ cells for antibody-targeted immunofluorescence assay for confocal/fluorescence microscopy. Key features • The protocol includes a simple method for preparing single testicular cells for immunofluorescence analysis. • Visualization of components in the manchette and sperm flagella using specific antibody markers.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 12","pages":"e5340"},"PeriodicalIF":1.0,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12254586/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144628079","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":"Surface Plasmon Resonance for the Interaction of Capsular Polysaccharide (CPS) With KpACE.","authors":"Zhe Wang, Lijun Wang, Xiaomin Zhang, Jing-Ren Zhang, Chao Cai","doi":"10.21769/BioProtoc.5346","DOIUrl":"10.21769/BioProtoc.5346","url":null,"abstract":"<p><p>The study of carbohydrate-protein interactions is crucial for clarifying biological processes and identifying potential drug candidates. However, due to the complex structure of carbohydrates, such as high molecular weight, dynamic flexibility, and high solution viscosity, it is challenging to study their interactions with diverse proteins. Conventional analytical techniques like isothermal titration calorimetry (ITC), X-ray crystallography, molecular dynamics (MD) simulations, and nuclear magnetic resonance (NMR) spectroscopy have limitations in revealing these molecular interactions. Surface plasmon resonance (SPR), an advanced optical biosensor technique, overcomes these limitations. It enables real-time, label-free monitoring of the interaction dynamics between carbohydrates and proteins through a continuous flow over a chip surface. In this study, we utilized SPR-based techniques to explore the interaction of capsular polysaccharides (CPS) of <i>Klebsiella pneumoniae</i> and the enzyme KpACE (<i>K. pneumoniae</i> acetylated capsule esterase). Our SPR-based analytical platform has several advantages, including shorter experimental time, a simulated physiological state, and minimal sample requirements for investigating carbohydrate-protein interactions. This approach expands the applicability scope of SPR technology and provides a valuable tool for a wide range of research. By using SPR, we successfully verified that KpACE acts on the acetyl groups of CPS, demonstrating its enzymatic activity, which is crucial for understanding the pathogenic mechanism of <i>K. pneumoniae</i> and developing potential antibacterial drugs. Key features • Conduct rapid screening of carbohydrate-protein interactions to determine binding affinity (KD). • Perform a comprehensive binding assay to assess the interactions between capsular polysaccharides (CPS) and mutant enzymes, thereby validating their catalytic sites. • Apply the methodology to achieve a highly sensitive, label-free, simulated physiological environment for substrate-enzyme interaction studies.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 12","pages":"e5346"},"PeriodicalIF":1.0,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12254591/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144628080","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":"A Hybrid 2D/3D Approach for Neural Differentiation Into Telencephalic Organoids and Efficient Modulation of FGF8 Signaling.","authors":"Michele Bertacchi, Gwendoline Maharaux, Michèle Studer","doi":"10.21769/BioProtoc.5354","DOIUrl":"10.21769/BioProtoc.5354","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Human brain development relies on a finely tuned balance between the proliferation and differentiation of neural progenitor cells, followed by the migration, differentiation, and connectivity of post-mitotic neurons with region-specific identities. These processes are orchestrated by gradients of morphogens, such as FGF8. Disruption of this developmental balance can lead to brain malformations, which underlie a range of complex neurodevelopmental disorders, including epilepsy, autism, and intellectual disabilities. Studying the early stages of human brain development, whether under normal or pathological conditions, remains challenging due to ethical and technical limitations inherent to working with human fetal tissue. Recently, human brain organoids have emerged as a powerful in vitro alternative, allowing researchers to model key aspects of early brain development while circumventing many of these constraints. Unlike traditional 2D cultures, where neural progenitors and neurons are grown on flat surfaces, 3D organoids form floating self-organizing aggregates that better replicate the cellular diversity and tissue architecture of the developing brain. However, 3D organoid protocols often suffer from significant variability between batches and individual organoids. Furthermore, few existing protocols directly manipulate key morphogen signaling pathways or provide detailed analyses of the resulting effects on regional brain patterning. • To address these limitations, we developed a hybrid 2D/3D approach for the rapid and efficient induction of telencephalic organoids that recapitulate major steps of anterior brain development. Starting from human induced pluripotent stem cells (hiPSCs), our protocol begins with 2D neural induction using small-molecule inhibitors to achieve fast and homogenous production of neural progenitors (NPs). After dissociation, NPs are reaggregated in Matrigel droplets and cultured in spinning mini-bioreactors, where they self-organize into neural rosettes and neuroepithelial structures, surrounded by differentiating neurons. Activation of the FGF signaling pathway through the controlled addition of FGF8 to the culture medium will modulate regional identity within developing organoids, leading to the formation of distinct co-developing domains within a single organoid. Our protocol combines the speed and reproducibility of 2D induction with the structural and cellular complexity of 3D telencephalic organoids. The ability to manipulate signaling pathways provides an additional opportunity to further increase system complexity, enabling the simultaneous development of multiple distinct brain regions within a single organoid. This versatile system facilitates the study of key cellular and molecular mechanisms driving early human brain development across both telencephalic and non-telencephalic areas. Key features • This protocol builds on the method established by Chambers et al. [1] for generating 2D neural progenitors","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 12","pages":"e5354"},"PeriodicalIF":1.0,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12222629/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144577223","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}