Current protocols最新文献

{"title":"Creating Optimal Western Blot Conditions for OPA1 Isoforms in Skeletal Muscle Cells and Tissue","authors":"Margaret Mungai,&nbsp;Amber Crabtree,&nbsp;Han Le,&nbsp;Johnathan Moore,&nbsp;Desiree Nguyen,&nbsp;Benjamin Rodriguez,&nbsp;Chanel Harris,&nbsp;Dominique C. Stephens,&nbsp;Heather K. Beasley,&nbsp;Edgar Garza-Lopez,&nbsp;Kit Neikirk,&nbsp;Bryanna Shao,&nbsp;Ashton Oliver,&nbsp;Genesis Wilson,&nbsp;Serif Bacevac,&nbsp;Larry Vang,&nbsp;Zer Vue,&nbsp;Neng Vue,&nbsp;Andrea G. Marshall,&nbsp;Kyrin Turner,&nbsp;Elma Zaganjor,&nbsp;Jianqiang Shao,&nbsp;Sandra Murray,&nbsp;Jennifer A. Gaddy,&nbsp;Celestine Wanjalla,&nbsp;Jamaine Davis,&nbsp;Steven M. Damo,&nbsp;Lori D. Banks,&nbsp;Antentor Hinton Jr","doi":"10.1002/cpz1.70004","DOIUrl":"10.1002/cpz1.70004","url":null,"abstract":"<p>OPA1 is a dynamin-related GTPase that modulates mitochondrial dynamics and cristae integrity. Humans carry eight different isoforms of OPA1 and mice carry five, all of which are expressed as short- or long-form isoforms. These isoforms contribute to OPA1's ability to control mitochondrial energetics and DNA maintenance. However, western blot isolation of all long and short isoforms of OPA1 can be difficult. To address this issue, we developed an optimized western blot protocol based on improving running time to isolate five different isoforms of OPA1 in mouse cells and tissues. This protocol can be applied to study changes in mitochondrial structure and function. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Western Blot Protocol for Isolating OPA1 Isoforms in Mouse Primary Skeletal Muscle Cells</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70004","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143191544","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":"Analyzing Pathophysiology and Immune Cells and Their Cytokines and Mediators in Precision-Cut Slices of the Murine Lung","authors":"Michaela Tatcheff,&nbsp;Christine Carvalho,&nbsp;Jonas Willar,&nbsp;Elvedina Nendel,&nbsp;Susanne Krammer,&nbsp;Mircea T. Chiriac,&nbsp;Shuting Zhou,&nbsp;Carol I. Geppert,&nbsp;Susetta Finotto","doi":"10.1002/cpz1.70087","DOIUrl":"10.1002/cpz1.70087","url":null,"abstract":"<p>Understanding the dynamic pathophysiology of diseases in the lung, such as asthma and chronic asthma, chronic obstructive pulmonary disease, and lung cancer, is crucial for the treatment, analysis, and outcome of these diseases. Unlike other traditional models, we suggest a protocol that is sustainable and reproducible and offers different analysis methods while maintaining <i>in vivo</i> lung architecture and immune dynamics. This protocol allows one to study the pathophysiological changes, including changes to the immune cells, cytokines, and mediators, in 30 precision-cut lung slices from a single murine lung. To accomplish this, the murine lung is infused with 2.5% low-melting-point agarose and is precision-cut-sliced. Our method also supports cell culture in refined medium and stimulation with clinically relevant stimuli, which helps to clarify the mechanisms of the disease. Evaluation of the samples and their supernatant includes multiplex assays, ELISA, histology, and immunohistochemistry. Additional sections are used to extract RNA for quantitative real-time PCR and RNA sequencing and/or other selected analysis, like flow cytometry. Using this method, we obtained murine lung slices that preserve the pathophysiology of the disease and allow a comprehensive analysis unlike other already-existing protocols. By retaining the dynamic immune mechanisms, we are able to see the histological damage caused by each disease. The results of this protocol can be used to improve our understanding and therapy options. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Obtaining precision-cut lung slices (PCLSs) from the murine lung</p><p><b>Support Protocol 1</b>: LL/2 cell treatment with G-418 solution</p><p><b>Support Protocol 2</b>: Murine model of lung adenocarcinoma and <i>in vivo</i> imaging</p><p><b>Support Protocol 3</b>: H&amp;E staining of PCLS sections</p><p><b>Support Protocol 4</b>: Measuring cytokines by ELISA</p><p><b>Support Protocol 5</b>: Measuring cytotoxic activity in PCLS conditioned medium</p><p><b>Support Protocol 6</b>: Analysis of RNA from PCLSs by real-time PCR</p><p><b>Support Protocol 7</b>: Flow cytometry analysis of cells isolated from PCLSs</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11773432/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143054653","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":"Correction: A Human Neuron/Astrocyte Co-culture to Model Seeded and Spontaneous Intraneuronal Tau Aggregation","authors":"Kevin Llewelyn Batenburg,&nbsp;Susan Karijn Rohde,&nbsp;Paulien Cornelissen-Steijger,&nbsp;Nicole Breeuwsma,&nbsp;Vivi Majella Heine,&nbsp;Wiep Scheper","doi":"10.1002/cpz1.70102","DOIUrl":"10.1002/cpz1.70102","url":null,"abstract":"<p><i>Current Protocols</i> is issuing corrections for the following protocol article.</p><p>Batenburg, K. L., Rohde, S. K., Cornelissen-Steijger, P., Breeuwsma, N., Heine, V. M., &amp; Scheper, W. (2023). A human neuron/astrocyte co-culture to model seeded and spontaneous intraneuronal tau aggregation. <i>Current Protocols</i>, <i>3</i>, e900. doi: 10.1002/cpz1.900</p><p>The current version online now includes these corrections and may be considered the authoritative version of record.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70102","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143054656","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":"Reproductive Risk Estimation Calculator for Balanced Translocation Carriers: An Improved Web-based Resource for Medical Genetics Professionals","authors":"Carolyn Trunca,&nbsp;Nancy R. Mendell,&nbsp;Samantha L. P. Schilit","doi":"10.1002/cpz1.70083","DOIUrl":"10.1002/cpz1.70083","url":null,"abstract":"<p>Balanced translocation carriers experience elevated reproductive risks, including pregnancy loss and children with anomalies due to generating chromosomally unbalanced gametes. While understanding the likelihood of producing unbalanced conceptuses is critical for individuals to make reproductive decisions, risk estimates are difficult to obtain as most balanced translocations are unique. To improve reproductive risk estimates, Drs. Trunca and Mendell created models based on a logistic regression analysis of a dataset of over 6000 individuals from over 1000 translocation families. While risk assessments using these models have been offered as a free service for years, this protocol aims to create a sustainable model for genetics professionals to obtain risk estimates for their patients directly. This protocol guides the user through collecting clinical information, using a risk-generating calculator based on the models, and interpreting the calculator outputs. This version of the protocol has been updated from the initial publication to introduce an additional resource to the community that is intended to improve accessibility and reduce error in calculating risks. In addition to the previously offered custom Java program, a web-based calculator that calculates age-adjusted miscarriage risks is now available. A practice tutorial is provided for both versions of the calculator to ensure competency in interpretation prior to use. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Estimation of reproductive risks for balanced translocation carriers</p><p><b>Basic Protocol 2</b>: Practical examples of typical patient encounters with instructive interpretations</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143049044","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}

{"title":"Quantifying Competitive Fitness in Yeast with High-Throughput Fluorescence Microscopy Imaging","authors":"Aruni S. Sumanarathne,&nbsp;Aleeza C. Gerstein","doi":"10.1002/cpz1.70093","DOIUrl":"10.1002/cpz1.70093","url":null,"abstract":"<p>Competitive fitness is a fundamental concept in evolutionary biology that captures the ability of organisms to survive, reproduce, and compete for resources in their environment. Competitive fitness is typically assessed in the lab by growing two or more competitors together and measuring the frequency of each at multiple time points. Traditional microbial competitive fitness assays are labor intensive and involve plating on solid medium and counting colonies. Here, we describe a method to quantitatively measure competitive fitness using fluorescence microscopic imaging and machine-learning-enabled image analysis to directly count the number of cells from each competitor in the mixed population. This high-throughput, primarily automated, and efficient process gives accurate and reproducible results for competitive fitness. Here, we describe the entire process, from sample preparation through microscopy to quantification, and provide instructions and scripts for the image analysis, fitness calculations, and sample data visualizations. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Sample preparation</p><p><b>Basic Protocol 2</b>: Photographing fluorescing and non-fluorescing cells using an EVOS microscope</p><p><b>Basic Protocol 3</b>: Counting fluorescing and non-fluorescing cells with Orbit Image Analysis</p><p><b>Basic Protocol 4</b>: Getting the average cell counts per well and changing the file names</p><p><b>Basic Protocol 5</b>: Calculating competitive fitness using R</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11771579/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143048612","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":"Plasmid Library Construction From Genomic DNA","authors":"Valeria Florez-Cardona,&nbsp;Jessica Khani,&nbsp;Emily McNutt,&nbsp;Bruno Manta,&nbsp;Mehmet Berkmen","doi":"10.1002/cpz1.70088","DOIUrl":"10.1002/cpz1.70088","url":null,"abstract":"<p>Functional genomic approaches have been effective at uncovering the function of uncharacterized genes and identifying new functions for known genes. Often these approaches rely on an <i>in vivo</i> screen or selection to associate genes with a phenotype of interest. These selections and screens are dependent upon the expression of proteins encoded in genomic DNA from an expression vector, such as a plasmid. Despite the utility of genomic DNA plasmid libraries, the protocols for their construction have remained unchanged in the past 40 years. Here, we present a procedure for constructing plasmid libraries from genomic DNA. This procedure is scalable and relies on simple techniques and common laboratory equipment and reagents. Briefly, the genomic DNA is extracted and then physically fragmented with a g-TUBE, overhangs are repaired, and fragments are selectively purified with magnetic beads to obtain an average fragment size of 2.5 kb. Blunted fragments are ligated into a blunt-end-digested and dephosphorylated vector. Finally, the library is amplified by electroporating the ligation into a high-transformation-efficiency <i>Escherichia coli</i> strain and extracting the plasmid DNA from the transformants. As a proof of concept, we built and sequenced three genomic libraries from different genomes and calculated their coverage using a next-generation sequencing (NGS) workflow. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Plasmid library construction</p><p><b>Alternate Protocol</b>: Selection of gDNA fragments using SageELF gel fractionator</p><p><b>Support Protocol 1</b>: Extraction of gDNA with phenol/chloroform</p><p><b>Support Protocol 2</b>: Vector preparation</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11752411/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143018604","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":"Modeling Malignant Mesothelioma in Genetically Engineered Mice","authors":"Yuwaraj Kadariya,&nbsp;Eleonora Sementino,&nbsp;Xiang Hua,&nbsp;Dietmar J. Kappes,&nbsp;Joseph R. Testa","doi":"10.1002/cpz1.70086","DOIUrl":"10.1002/cpz1.70086","url":null,"abstract":"<p>Mesothelioma is a lethal cancer of the serosal lining of the body cavities. Risk factors include environmental and genetic factors. Asbestos exposure is considered the principal environmental risk factor, but other carcinogenic mineral fibers, such as erionite, also have a causal role. Pathogenic germline (heritable) mutations of specific genes, especially <i>BAP1</i>, are thought to predispose the individual to mesothelioma in about 10% of cases. Somatic mutations and deletions of specific tumor suppressor genes, particularly <i>BAP1</i>, <i>CDKN2A/B</i>, and <i>NF2</i>, occur frequently in human mesothelioma, and asbestos-exposed mice with heterozygous deletions of any one of these genes have been shown to develop mesothelioma more often and at an accelerated rate than in control animals. Autochthonous mesothelioma mouse models, which are genetically engineered to carry multiple genetic lesions matching those observed in the human disease counterpart, closely resemble the disease phenotype and the extensive inflammatory responses that characterize human mesothelioma. Because autochthonous mice do not require asbestos exposure and form tumors rapidly, these models are invaluable for assessing novel therapeutic strategies in an immunocompetent setting. The overlapping genetic, epigenetic, and immune environments of the tumors observed in these genetically engineered mouse models (GEMMs) and human primary mesothelioma specimens support the clinical relevance of these preclinical models. This article presents protocols for studies of asbestos-induced mesothelioma in GEMMs and non-carcinogenic conditional knockout models of mesothelioma, including an example of a preclinical application. These models are invaluable for understanding the biological underpinnings of mesothelioma and for testing new therapeutics and chemoprevention or interception agents. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Generation of a genetically engineered mouse model (GEMM) with a germline <i>Bap1</i> knockout allele</p><p><b>Basic Protocol 2</b>: Generation of GEMMs with germline <i>Bap1</i> knock-in alleles</p><p><b>Basic Protocol 3</b>: Asbestos carcinogenicity investigations with GEMMs</p><p><b>Basic Protocol 4</b>: Preclinical chemoprevention and chemotherapy studies using a GEMM with asbestos-induced mesothelioma</p><p><b>Basic Protocol 5</b>: Generation of a GEMM with conditional knockout of <i>Bap1</i></p><p><b>Basic Protocol 6</b>: Generation of a conditional knockout model of mesothelioma</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142960348","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}

{"title":"A Simple Protocol for Visualization of RNA–Protein Complexes by Atomic Force Microscopy","authors":"Andrea Tripepi,&nbsp;Huma Shakoor,&nbsp;Petr Klapetek","doi":"10.1002/cpz1.70084","DOIUrl":"10.1002/cpz1.70084","url":null,"abstract":"<p>Atomic force microscopy (AFM) has recently received increasing interest in molecular biology. This technique allows quick and reliable detection of biomolecules. However, studying RNA–protein complexes using AFM poses significant challenges. Here, we describe a simple and reliable method to visualize positively charged proteins bound to RNA that does not require metallic cations. This method allowed us to effectively detect and visualize Staufen–RNA complexes by height or logarithmic stiffness. The study of the mechanical properties is particularly important in the case of protein-coated RNA complexes, where RNA cannot be detected by height channel. In any case, it is necessary to compare AFM data with the data derived from other techniques like nuclear magnetic resonance, X-ray crystallography, cryogenic electron microscopy, and small-angle X-ray scattering. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Preparation and visualization of RNA–protein complex</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11713223/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142960698","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":"Miniscope Imaging of Nucleus Accumbens Neural Activity in Freely Behaving Rats: Virus Injection, Gradient Index Lens Implantation, Recording Strategies, and Analytical Methods","authors":"Nicholas J. Beacher,&nbsp;Michael W. Wang,&nbsp;Matthew C. Broomer,&nbsp;Jessica Y. Kuo,&nbsp;Kayden A. Washington,&nbsp;Miranda Targum,&nbsp;Yan Zhang,&nbsp;Giovanni Barbera,&nbsp;Da-Ting Lin","doi":"10.1002/cpz1.70090","DOIUrl":"10.1002/cpz1.70090","url":null,"abstract":"<p><i>In vivo</i> calcium imaging in freely moving rats using miniscopes provides valuable information about the neural mechanisms of behavior in real time. A gradient index (GRIN) lens can be implanted in deep brain structures to relay activity from single neurons. While such procedures have been successful in mice, few reports provide detailed procedures for successful surgery and long-term imaging in rats, which are better suited for studying complex human behaviors. We present a robotic surgical protocol for same-day virus injection and GRIN lens implantation into the rat nucleus accumbens core. Our procedure utilizes a direct lens insertion without tissue aspiration and produces quality image retention for months of recording. We also describe daily recording strategies to minimize damage and promote long-term imaging. Finally, we present custom protective strategies to eliminate the need to remove miniscopes between sessions. This methodology protects rats from repeated isoflurane exposure and ensures a consistent focal plane for the entirety of the experiment. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Craniotomy</p><p><b>Basic Protocol 2</b>: Virus injection</p><p><b>Basic Protocol 3</b>: GRIN lens implantation</p><p><b>Basic Protocol 4</b>: Baseplate mounting and assessment of the anesthetized rat</p><p><b>Basic Protocol 5</b>: Assessment of the awake, behaving rat</p><p><b>Support Protocol 1</b>: Protective miniscope cone fabrication</p><p><b>Support Protocol 2</b>: Miniscope cable fabrication</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11718238/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142960329","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":"Generation of Retinal Ganglion Cells from Reprogrammed Keratocytes of Non-Glaucoma and Glaucoma Donors","authors":"Shahna S. Hameed,&nbsp;Tasneem P. Sharma","doi":"10.1002/cpz1.70091","DOIUrl":"10.1002/cpz1.70091","url":null,"abstract":"<p>Human induced pluripotent stem cell (hiPSC)-based disease modeling can be successfully recapitulated to mimic disease characteristics across various human pathologies. Glaucoma, a progressive optic neuropathy, primarily affects the retinal ganglion cells (RGCs). While multiple groups have successfully generated RGCs from non-diseased hiPSCs, producing RGCs from glaucomatous human samples holds significant promise for understanding disease pathology by revealing patient-specific disease signatures. Given that keratocytes originate from the neural crest and previous reports suggest that ocular fibroblasts from glaucomatous donors carry pathogenic signatures, it is highly plausible that these signatures imprinted within the keratocytes will also be present in the derived RGCs. Thus, we aimed to generate RGCs from both glaucomatous and non-glaucomatous donor keratocytes and validate disease-specific signatures in 3D retinal organoids and in isolated RGCs. Our protocol describes the generation of iPSCs from keratocytes of both glaucomatous and non-glaucomatous donors, followed by their differentiation into retinal organoids. Subsequent isolation and culturing of RGCs were performed. Disease signatures in the RGCs were validated in both 3D retinal organoids (ROs) and 2D RGC cultures, and glaucomatous RGCs in 3D and 2D cultures demonstrated increased cleaved CASP3 and significant RGC loss, indicating disease imprints in the hiPSC-derived RGCs. This model offers a venue and high throughput platform for studying glaucomatous disease pathology and holds significant potential for drug discovery using RGCs derived from human donors. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Culturing of keratocytes from human cadaveric donors</p><p><b>Basic Protocol 2</b>: Reprogramming donor keratocytes into iPSCs</p><p><b>Basic Protocol 3</b>: Evaluation of chromosomal loss during reprogramming in iPSCs by karyotyping</p><p><b>Basic Protocol 4</b>: Generation of 3D ROs</p><p><b>Basic Protocol 5</b>: Dissociation and culturing of RGCs from 3D ROs</p><p><b>Support Protocol 1</b>: Immunostaining for phenotypic characterization of cells</p><p><b>Support Protocol 2</b>: Sectioning of 3D ROs and immunostaining</p><p><b>Support Protocol 3</b>: Western blotting for cleaved CASP3 and THY1</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11713219/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142960699","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}