Bio-protocol最新文献

{"title":"Efficient Gene Knockdown in Adult Zebrafish Retina by Intravitreal Injection.","authors":"Eyad Shihabeddin, Abirami Santhanam, Stephan Tetenborg, Alexandra L Aronowitz, John O'Brien","doi":"10.21769/BioProtoc.5436","DOIUrl":"10.21769/BioProtoc.5436","url":null,"abstract":"<p><p>High-throughput sequencing has created a tremendous amount of information about the genes expressed in various cell types and tissues throughout the body. As such, there is a need for a quick and effective method to knock down genes of interest in order to investigate their roles. While there are many approaches for this in mammalian models, there are limited ways to knock down genes of interest in adult zebrafish. Unlike mammals, zebrafish have the natural ability to regenerate their neurons after injury or disease is detected, making them a staple in regenerative studies. Unfortunately, current approaches for gene knockdown in the retina of adult zebrafish are costly and provide a barrier for many scientists. We provide two cost-effective approaches for targeted gene knockdowns in adult zebrafish retinas. We describe this approach through the use of Vivo-morpholinos and lipid-encapsulated siRNAs that target the expression of the proliferating cell nuclear antigen (<i>PCNA</i>) gene in adult zebrafish. We also describe how to collect and process retina samples for downstream immunohistochemistry, imaging, and quantification. Overall, this protocol will provide researchers with a straightforward, cheap, and effective method to perform targeted gene knockdowns in adult zebrafish retinas. Key features • This protocol provides researchers with an approach to knock down genes of interest in adult zebrafish retina without the use of electroporation. • This protocol can be performed without causing an acute damage response in the retina. • This protocol allows targeting of genes in both proliferating cells and terminally differentiated cells. • This protocol allows the retina to be collected and processed for further downstream analysis.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 17","pages":"e5436"},"PeriodicalIF":1.1,"publicationDate":"2025-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12423274/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145066739","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":"Simultaneous RNA Fluorescent In Situ Hybridization and Immunofluorescent Staining of Mouse Muscle Stem Cells on Fresh Frozen Skeletal Muscle Sections.","authors":"Vedant R Lakkundi, Maria L Perez, Kartik Soni, Albert E Almada","doi":"10.21769/BioProtoc.5435","DOIUrl":"10.21769/BioProtoc.5435","url":null,"abstract":"<p><p>Adult muscle stem cells (MuSCs) are the key cellular source for regenerating skeletal muscle in vertebrates. MuSCs are typically identified in skeletal muscle by the expression of the paired box protein 7 (PAX7) protein. Here, we developed a combined RNA fluorescent in situ hybridization (FISH) using RNAscope technology and an immunofluorescence (IF) protocol for the simultaneous detection of <i>Pax7</i> mRNA and PAX7 protein in individual MuSCs in vivo. Interestingly, we show that while most PAX7⁺ (protein) MuSCs express <i>Pax7</i> mRNA, there is a subset of <i>Pax7</i> ⁺ (mRNA) cells that do not express PAX7 protein. Altogether, we developed a combined FISH/IF protocol that allows for the co-detection of mRNA and protein in MuSCs in vivo, a strategy that can be applied to any target gene. The functional significance of the <i>Pax7</i>-expressing subset of cells lacking PAX7 protein prior to injury remains unknown. Key features • Extensive step-by-step details for an optimized protocol combining traditional immunofluorescence with RNA fluorescent in situ hybridization (FISH) using ACDBio's RNAscope technology. • Allows for the co-detection of protein and mRNA in muscle stem cells (MuSCs) in mouse skeletal muscle tissue in vivo in ~2 days. • Validation of our protocol uncovers a subset of cells expressing <i>Pax7</i> mRNA but not PAX7 protein.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 17","pages":"e5435"},"PeriodicalIF":1.1,"publicationDate":"2025-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12423284/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145066849","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":"Ultrafast Isolation of Synaptic Terminals From Rat Brain for Cryo-Electron Tomography Analysis.","authors":"Rong Sun, Qiangjun Zhou","doi":"10.21769/BioProtoc.5429","DOIUrl":"10.21769/BioProtoc.5429","url":null,"abstract":"<p><p>Understanding the nanoscale organization and molecular rearrangement of synaptic components is critical for elucidating the mechanisms of synaptic transmission and plasticity. Traditional synaptosome isolation protocols involve multiple centrifugation and resuspension steps, which may cause structural damage or alter the synaptosomal fraction, compromising their suitability for cryo-electron tomography (cryo-ET). Here, we present an ultrafast isolation method optimized for cryo-ET that yields two types of synaptosomal fractions: synaptosomes and synaptoneurosomes. This streamlined protocol preserves intact postsynaptic membranes apposed to presynaptic active zones and produces thin, high-quality samples suitable for in situ structural studies. The entire procedure, from tissue homogenization to vitrification, takes less than 15 min, offering a significant advantage for high-resolution cryo-ET analysis of synaptic architecture. Key features • Ultrafast synaptic terminal isolation from tissue homogenization to vitrification completed within 15 min. • Retention of postsynaptic membranes with synaptic receptors and postsynaptic density (PSD) proteins. • The thickness of the samples is suitable for in situ cryo-ET analysis. • Enables cryo-ET studies of synaptic structures and postsynaptic membrane proteins such as AMPA receptors.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 17","pages":"e5429"},"PeriodicalIF":1.1,"publicationDate":"2025-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12423272/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145066183","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 Simple and Adaptable Method for Cloning Genes Into Transposon Vectors Using Topo and Restriction Systems for Chicken Embryo Transgenesis.","authors":"Pamela Kirimi, Noah Okumu, John M Maingi, Joseph Ngeranwa, Philip Nyaga, Yatinder Binepal","doi":"10.21769/BioProtoc.5416","DOIUrl":"10.21769/BioProtoc.5416","url":null,"abstract":"<p><p>Transposon-based genetic transformation enables stable transgene integration in avian genomes and is increasingly used in the development of transgenic chickens for enhanced disease resistance, productivity, and biopharmaceutical applications. Conventional transformation techniques in avian biotechnology, including viral vectors and primordial germ cell (PGC) manipulation, are limited by biosafety risks, low efficiency, and technical complexity. This protocol outlines a two-step cloning approach for generating transposon-compatible gene constructs suitable for chicken embryo microinjection. Topoisomerase-based (TOPO) cloning is used as the first step due to its ability to directly clone PCR-amplified products without the need for restriction site-engineered primers while simultaneously producing an insert flanked with EcoRI restriction sites. The insert is subsequently transferred into the transposon vector through EcoRI-mediated restriction digestion and ligation. This approach simplifies construct generation by integrating the speed of TOPO cloning with the precision of restriction cloning, while ensuring compatibility with transposon-mediated integration systems. The protocol is efficient, reproducible, and does not require specialized equipment, providing a practical and scalable tool for gene construct assembly in avian transgenesis research. Key features • Uses TOPO PCR cloning for initial gene insertion. • Applies restriction cloning to transfer inserts to the destination vector. • Employs pKTol2C-EGFP as the final transposon vector. • Suitable for generating constructs for chicken embryo transgenesis.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 16","pages":"e5416"},"PeriodicalIF":1.1,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12378422/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144982004","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":"ClearDepth Method for Evaluations of Root Depth in Soil-Filled Pots.","authors":"Michel Ruiz Rosquete, Juan Gonzalez, Natalie Gonzalez, Kristen Wertz, Suyash Patil, Wolfgang Busch","doi":"10.21769/BioProtoc.5425","DOIUrl":"10.21769/BioProtoc.5425","url":null,"abstract":"<p><p>Despite its significant relevance to drought adaptation, optimization of nutrient acquisition, and carbon sequestration in soil, genetic factors determining root depth remain poorly explored, mostly due to the limitations of the methods currently available to estimate it. Although several such methods have been developed for crops, their applicability to large-scale studies and those involving smaller, more fragile root systems is severely limited. To address this, we have developed ClearDepth, a simple, non-destructive, low-cost method. In ClearDepth, the root system develops naturally inside the soil in clear pots. As it expands, secondary roots reach the transparent walls of the pot (\"wall roots\"), becoming visible. The shallowness of each wall root is then measured (wall root shallowness, WRS), and the depth of the root system is expressed as the average of all single WRS measurements. We demonstrated the suitability of ClearDepth for root depth studies using <i>Arabidopsis thaliana</i> and <i>Oryza sativa</i> (rice), two species with contrasting root system architecture (RSA) and root size. The robustness and sensitivity of the WRS trait allow us not only to reproducibly discriminate between shallow and deep root systems but also to detect smaller yet significant differences in depth determined by the influence of environmental factors, such as light. Here, we present a comprehensive protocol for utilizing this method. Key features • ClearDepth measures the depth of a minimum number of secondary roots, set by the user, to estimate the depth of the root system. • The method captures differences of root depth at a spatio-developmental stage rather than at one specific time point after planting. • ClearDepth captures differences in root depth independently of differences in total root biomass.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 16","pages":"e5425"},"PeriodicalIF":1.1,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12378426/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144982296","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":"Rapid Isolation and Flow Cytometry Analysis of Murine Intestinal Immune Cells After Chemically Induced Colitis.","authors":"Ashish K Singh, Alfonso Blanco, Ray Sinnott, Ulla G Knaus","doi":"10.21769/BioProtoc.5417","DOIUrl":"10.21769/BioProtoc.5417","url":null,"abstract":"<p><p>Chemically induced murine colitis models are widely used to understand intestinal homeostasis and inflammatory responses during acute and chronic gut inflammation, such as inflammatory bowel disease (IBD). Resident populations of immune cells, together with those recruited during an inflammatory response, maintain intestinal immunity by mounting an effective immune response to enteropathogenic microbes while at the same time maintaining tolerance against commensals. To better understand the disease mechanism, studying different immune cell populations and their dynamic changes during infection and inflammation is essential. However, isolating healthy and viable immune populations, particularly hyperactivated neutrophils and macrophages from the inflamed gut (i.e., active disease site), is challenging as tissues are usually subjected to rigorous enzymatic digestion for an extended period. Here, we describe a method that uses a cell dissociator (Medimachine II from Syntec International) to separate intestinal tissue after short enzymatic digestion to obtain a single-cell suspension. This technique facilitates the isolation of immune cells from mouse intestinal tissues in high quantity and with superior viability in a very short time frame. This protocol delivers 80%-90% cell viability, which is 1.5 to 2-fold higher than conventional methods of isolating cells from inflamed mouse colons. The composition, phenotype, activation state, and gene expression profile of cells isolated using this protocol can be assessed by using multiple methods, including, but not limited to, flow cytometry, quantitative PCR, immunoblotting, mass spectrometry, single-cell RNA sequencing, and functional readouts such as reactive oxygen species (ROS) production. Key features • Infiltrating immune cells are key drivers of intestinal inflammation. • Isolating viable cells from the inflamed colon is slow and may alter cell activation and expression profiles. • This protocol enables faster isolation with improved cell viability. • Isolated cells can be further purified using magnetic beads or flow cytometry for downstream analysis.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 16","pages":"e5417"},"PeriodicalIF":1.1,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12378419/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144982304","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":"Production of Homogeneous, Functional Zinc-Finger Arrays in High Yield With Two Chromatographic Steps.","authors":"Jingchang Liang, Maia Azubel, Guanqiao Wang, Yan Nie, Roger D Kornberg, Andrew J Beel, Pierre-Jean Matteï","doi":"10.21769/BioProtoc.5420","DOIUrl":"10.21769/BioProtoc.5420","url":null,"abstract":"<p><p>Zinc-finger (ZF) arrays are compact, sequence-specific polynucleotide-binding domains, which have been used to target the delivery of diverse effector domains, enabling applications such as gene identification, localization, regulation, and editing. To facilitate in vitro applications of ZF arrays, we have developed a general method for their expression and purification. Here, we describe a protocol involving two chromatographic steps that yields homogeneous and functional ZF arrays in milligram quantities. Key features • A general method for expressing and purifying C2H2 ZF arrays in <i>E. coli</i>, compatible with both natural and artificial ZFs. • The His-SUMO tag improves ZF-array solubility, eliminating the need for denaturation and refolding steps. • Simple, two-step purification yields milligram-scale ZF arrays suitable for downstream applications.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 16","pages":"e5420"},"PeriodicalIF":1.1,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12378415/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144982301","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":"Transplantation of Cultured Myoblasts Into Intact Skeletal Muscle and Analysis of Muscle Contraction Force in Mice Model.","authors":"Kitora Dohi, Yasuro Furuichi","doi":"10.21769/BioProtoc.5413","DOIUrl":"10.21769/BioProtoc.5413","url":null,"abstract":"<p><p>Cell transplantation is a promising strategy for treating age-related muscle atrophy, but its critical application remains limited. Cultured myoblasts, unlike freshly isolated muscle stem cells, show poor engraftment efficiency and fail to contribute effectively to muscle regeneration. Moreover, successful engraftment generally requires prior muscle injury, as skeletal muscle regeneration is typically triggered by a damaged microenvironment. These limitations present major obstacles for applying cell therapy to sarcopenia, where muscle degeneration occurs without injury. In this protocol, we describe a novel approach that enables the transplantation of cultured myoblasts into intact skeletal muscle without the need for preexisting injuries or genetic modification. By combining myoblasts with extracellular matrices (ECM), such as Matrigel, which mimic the native muscle niche and support cell survival, adhesion, proliferation, and differentiation, we achieve efficient engraftment and increased muscle mass without the need for preexisting injury. The ECM also provides a scaffold and retains bioactive factors that enhance the regenerative capacity of transplanted cells. This is the first protocol that enables robust myoblast engraftment in non-injury muscle conditions, offering a practical tool for studying and potentially treating sarcopenia. Key features • Cultured myoblasts mixed with extracellular matrix components are transplanted into intact skeletal muscle. • Contraction force measurement of the tibialis anterior muscle in vivo. • Cell transplantation without muscle injury would be applied for the treatment of sarcopenia.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 16","pages":"e5413"},"PeriodicalIF":1.1,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12378423/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144982278","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":"Assessing Human Treg Suppression at Single-Cell Resolution Using Mass Cytometry.","authors":"Jonas Nørskov Søndergaard, Janyerkye Tulyeu, David Priest, Shimon Sakaguchi, James B Wing","doi":"10.21769/BioProtoc.5424","DOIUrl":"10.21769/BioProtoc.5424","url":null,"abstract":"<p><p>Regulatory T cells (Tregs) are essential for maintaining immune balance by controlling the activation and expansion of other immune cells. Conventional suppression assays often rely on co-culturing purified cell populations, which limits multiplexed phenotyping and physiological relevance. This protocol describes a high-dimensional, single-cell assay for profiling Treg-mediated suppression within a peripheral blood mononuclear cell (PBMC) system. Tregs are first isolated by cell sorting and then reintroduced into autologous PBMCs at defined ratios. A 52-marker mass cytometry (CyTOF) panel is used to quantify cell division and phenotypic responses across multiple immune subsets. This approach allows for integrated analysis of Treg function with broad compatibility for patient profiling and drug evaluation. Key features • Quantifies Treg-mediated suppression in autologous PBMCs at single-cell resolution. • Enables high-dimensional phenotyping and proliferation tracking across multiple immune subsets using a 52-marker CyTOF panel. • Maintains physiological relevance by assessing suppression in a complex PBMC environment. • Compatible with patient-derived samples and drug perturbation experiments for translational immunology applications.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 16","pages":"e5424"},"PeriodicalIF":1.1,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12378424/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144982287","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":"Isolation and Ex Vivo Testing of CD8⁺ T-Cell Division and Activation Using Mouse Splenocytes.","authors":"Melissa Dolan, Yuhao Shi, Amber McKenery, Stephanie Tzetzo, Kohei Chida, Kazuaki Takabe, Scott I Abrams, John M L Ebos","doi":"10.21769/BioProtoc.5423","DOIUrl":"10.21769/BioProtoc.5423","url":null,"abstract":"<p><p>This protocol describes an ex vivo co-culture method to assess CD8⁺ T-cell activation, proliferation, and cytotoxic potential using bulk splenocytes isolated from immunocompetent mice. Mouse splenocytes are stimulated with anti-CD3 and anti-CD28 antibodies to activate CD8⁺ T cells, which are then co-incubated with either cancer cells or cancer cell-derived conditioned media (CM) to evaluate tumor-driven modulation of immune cell functions. The use of unfractionated splenocytes preserves physiological cell-cell interactions, eliminating the need for exogenous interleukin (IL-2) and bypassing flow sorting, which simplifies the workflow and reduces experimental variability. CD8⁺ T-cell responses are measured via flow cytometry, using markers of proliferation (CFSE dilution), activation (CD69), and effector function (Granzyme B and IFNγ). Additionally, immune-mediated tumor cell death is evaluated by Annexin-V/7-AAD staining. Together, this experimental platform supports the investigation of both cell contact-dependent and contact-independent mechanisms of immune cell modulation in a cost-effective and reproducible setting. Key features • Enables isolation and stimulation of splenocytes to assess CD8⁺ T-cell responses to cancer cells or their secreted factors. • Supports evaluation of CD8⁺ T-cell activation, proliferation, and effector function by flow cytometry. • Allows functional assessment of tumor-driven suppression of T-cell activation in co-culture or conditioned media. • Measures cancer cell death resulting from interactions with activated CD8⁺ T cells in splenocyte co-cultures.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 16","pages":"e5423"},"PeriodicalIF":1.1,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12378427/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144982343","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}