Bio-protocol最新文献

{"title":"Constructing and Implementing a Low-Cost On-Demand Morris Water Maze Platform.","authors":"Bradley Stinnette, Jeffrey M Long, Craig Myrum","doi":"10.21769/BioProtoc.5428","DOIUrl":"10.21769/BioProtoc.5428","url":null,"abstract":"<p><p>The Morris water maze (MWM) is one of the most widely used procedures to assess hippocampus-dependent spatial learning and memory in rodents. By varying test protocols, researchers can test several different domains of learning and memory. Over multiple testing days, animals learn to swim to a platform hidden just under the water surface by using the spatial relationship between distal cues and the platform. Probe trials, where the platform is rendered unavailable, measure rodents' spatial bias for the area where the platform was previously located. The ability of researchers to control the availability of the platform \"on-demand\" offers both practical and methodological advantages. Despite MWM's prominence in the field of behavioral neuroscience, the high cost of purchasing a commercial MWM package is often prohibitively expensive for many research labs, especially on-demand platforms. Here, we describe a low-cost strategy for a build-your-own MWM that includes a remote-controlled on-demand platform (~530 USD) and tank (~550 USD). It is our hope that disseminating low-cost strategies aimed at expanding access to high-quality research tools at underfunded research institutions will accelerate biomedical discovery and foster further innovation. Key features • An on-demand platform allows for seamless testing (e.g., during probe trials, when the platform is usually removed), without interrupting the experiment to adjust/remove the platform. • The focus here is to offer a step-by-step, economical alternative to otherwise costly, commercial on-demand platforms. • Additionally, the protocol offers cost-effective ways of assembling the tank, preparing the testing environment, and implementing a testing protocol. • We include testing strategies specifically developed for aged rats, as these animals are routinely used in our laboratories.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 17","pages":"e5428"},"PeriodicalIF":1.1,"publicationDate":"2025-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12423283/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145066745","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":"NanoPDLIM2-Based Combination Therapy for Lung Cancer Treatment in Mouse Preclinical Studies.","authors":"Thi Hoa Le, Fan Sun, Gutian Xiao, Zhaoxia Qu","doi":"10.21769/BioProtoc.5437","DOIUrl":"10.21769/BioProtoc.5437","url":null,"abstract":"<p><p>This protocol describes the preparation, administration, and analysis of a nanoparticle-based therapeutic strategy (nanoPDLIM2) in combination with PD-1 immune checkpoint blockade immunotherapy and chemotherapy for the treatment of lung cancer in mouse preclinical studies. NanoPDLIM2 uses a polyethyleneimine (PEI)-based delivery system that encapsulates PDLIM2 expression plasmids for reconstituting PDLIM2 that is repressed in tumors. This approach induces tumor immunogenicity, suppresses drug resistance, and improves treatment efficacy when used in combination with carboplatin, paclitaxel, and anti-PD-1 antibodies. The protocol describes steps for mouse lung tumor induction, nanoPDLIM2 and other therapeutic reagents' preparation and administration, and subsequent analysis of tumor burden, immune response, and toxicity, providing a reproducible approach for investigators. Key features • Comprehensive workflow for preparation and delivery of nanoPDLIM2. • Combination of nanoPDLIM2 with PD-1 blockade and chemotherapeutics for superior efficacy in lung cancer treatment. • Detailed protocols for therapeutic reagents preparation, administration, tumor examination, immune analysis, health monitoring, and toxicity evaluation in a preclinical lung cancer model.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 17","pages":"e5437"},"PeriodicalIF":1.1,"publicationDate":"2025-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12423285/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145066752","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":"Novel Experimental Approach to Investigate Immune Control of Vascular Function: Co-culture of Murine Aortas With T Lymphocytes or Macrophages.","authors":"Taylor C Kress, Candee T Barris, Simone Kennard, Eric J Belin de Chantemèle","doi":"10.21769/BioProtoc.5440","DOIUrl":"10.21769/BioProtoc.5440","url":null,"abstract":"<p><p>Cardiovascular disease, the current leading cause of death worldwide, is a multifactorial disorder that involves a strong contribution of both the innate and adaptive immune systems. Overactivation of the immune system and inappropriate secretion of pro-inflammatory cytokines lead to vascular impairments and the development of cardiovascular disorders, including hypertension, atherosclerosis, and peripheral artery disease. Lymphocytes, macrophages, and dendritic cells can all secrete pro-inflammatory cytokines. This makes it challenging to isolate a specific subset of immune cells, particularly cytokines, and their contribution to vascular dysfunction remains difficult to elucidate. To solve this problem, our laboratory has developed the novel \"immune cell-aorta\" co-culture system described herein. This experimental protocol enables investigators to isolate an immune cell of interest and identify the cytokine(s) at the origin of vascular alterations. Key features • Novel ex vivo approach combining the culture of one population of immune cells with blood vessels. • No direct contact between the cells and the blood vessels; the model enables studying the role of immune cell-derived factors or cytokines on vascular function. • Blood vessels can subsequently be used for functional (wire/pressure myography), molecular (western blot, quantitative real-time RT-PCR), and histological studies.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 17","pages":"e5440"},"PeriodicalIF":1.1,"publicationDate":"2025-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12423277/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145066725","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":"Use of a High-Affinity Ubiquitin-Binding Domain to Detect and Purify Ubiquitinated Substrates and Their Interacting Proteins.","authors":"Nitu Saha, Mengwen Zhang, Mark Hochstrasser","doi":"10.21769/BioProtoc.5426","DOIUrl":"10.21769/BioProtoc.5426","url":null,"abstract":"<p><p>OtUBD is a high-affinity ubiquitin-binding domain (UBD) derived from a large protein produced by the microorganism <i>Orientia tsutsugamushi</i>. The following protocol describes a step-by-step process for the enrichment of ubiquitinated proteins from baker's yeast and mammalian cell lysates using OtUBD. The OtUBD affinity resin can strongly enrich both mono- and poly-ubiquitinated proteins from crude lysates. The protocol further describes the use of different buffer formulations to specifically enrich for proteins covalently modified by ubiquitin with or without proteins that associate with them. Combining different OtUBD-mediated enrichment protocols with liquid chromatography-tandem mass spectrometry (LC-MS/MS) helps distinguish the pool of covalently ubiquitinated proteins (the ubiquitinome) from ubiquitin- or ubiquitinated protein-interacting proteins (the ubiquitin interactome). The OtUBD tool described in the protocol has been used successfully with downstream applications such as immunoblotting and differential proteomics. It provides researchers with a versatile and economical tool for the study of ubiquitin biology. Key Features • The protocol offers a native workflow and a denaturing workflow for enrichment of ubiquitinated proteins with or without noncovalently associated proteins, respectively. • Included in the protocol are different resin compositions, lysate preparation methods, elution methods, and pulldown formats to suit different experimental needs. • The protocol has been used in various applications, including immunoblotting, proteomics, and UbiCREST (ubiquitin chain restriction), and works with all types of ubiquitin conjugates. • The protocol was developed and tested with budding yeast and mammalian cell lysates but can be adapted to other biological samples and organisms.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 17","pages":"e5426"},"PeriodicalIF":1.1,"publicationDate":"2025-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12423275/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145066263","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":"PhosphoLIMBO: An Easy and Efficient Protocol to Separate and Analyze Phospholipids by HPTLC From Plant Material.","authors":"Louise Fougère, Hortense Moreau, Cécile Mirande-Bret, Laetitia Fouillen, Yohann Boutté","doi":"10.21769/BioProtoc.5434","DOIUrl":"10.21769/BioProtoc.5434","url":null,"abstract":"<p><p>Phospholipids are major structural and regulatory elements of biological membranes and are involved in many different cellular and physiological processes. In this protocol, we provide an easy, cost-effective, and efficient method to obtain an overview of the phospholipid composition using high-performance thin layer chromatography (HPTLC). While the currently known phospholipid separation methods based on HPTLC display co-migration of certain lipid classes, the method we describe here allows the separation of all phospholipid classes, including anionic phospholipids in plant samples. This protocol combines elements of the classical Vitiello and Touchstone solvent systems to optimize phospholipid separation in a scaled pattern. Here, we provide a full characterization of this method, including statistical analyses of the retention factor of each phospholipid to show the robustness of the method and its efficiency in separating all phospholipid classes of a biological sample. Key features • Analysis of phospholipid composition through an easy, fast, robust, and cost-effective HPTLC method. • Separation of anionic phospholipids from the other phospholipid species. • Full overview of all phospholipids categories, including anionic phospholipids. • Qualitative and quantitative approach.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 17","pages":"e5434"},"PeriodicalIF":1.1,"publicationDate":"2025-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12423273/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145066684","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":"Time-Resolved cAMP Level Determination in Frog Retina Samples Using LC-MS/MS.","authors":"Olga V Chernyshkova, Mikhail V Belyakov, Darya A Meshalkina, Mikhail L Firsov","doi":"10.21769/BioProtoc.5431","DOIUrl":"10.21769/BioProtoc.5431","url":null,"abstract":"<p><p>The phototransduction cascade allows photoreceptors to detect light across a wide range of intensities without saturation, with cGMP serving as the second messenger and calcium feedback as the key regulatory mechanism. While experimental evidence suggests that cAMP may also play a role in modulating this cascade, such regulation would necessitate rapid changes in cAMP levels on a timescale of seconds. However, data on the dynamics of intracellular cAMP changes in photoreceptors remain scarce, primarily due to the limitations of conventional fluorescence-based methods in this specialized sensory system. To address this gap, we developed a methodology combining rapid cryofixation of retinal samples following light stimulation with the isolation of outer segment preparations. The rapid cryofixation setup comprises six computer-controlled sections, each with a high-speed stepper motor-driven lever that rapidly moves the specimen in a 180° arc within ~80 ms to press it against a liquid nitrogen-cooled copper cylinder for fixation. Using highly sensitive metabolomics techniques, we measured cAMP levels in these samples. This approach enables the investigation of rapid cAMP dynamics and its potential regulatory role in phototransduction, providing a foundation for understanding the interplay between cAMP and PKA signaling in photoreceptor function. Key features • The protocol provides ms time resolution in retina outer segment sampling in response to light stimulus with cryofixation, conserving proteome and metabolome response features. • The protocol allows direct cAMP quantification with an average level of 11.4 ± 0.5 pmol/mg of protein in the dark.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 17","pages":"e5431"},"PeriodicalIF":1.1,"publicationDate":"2025-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12423281/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145066860","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":"Real-Time Imaging of Specific Genomic Loci With CRISPR/dCas9 in Human Cells Using CRISPRainbow.","authors":"Thomas J Versosky, Dilshodbek U Nishonov, Li-Chun Tu","doi":"10.21769/BioProtoc.5432","DOIUrl":"10.21769/BioProtoc.5432","url":null,"abstract":"<p><p>Proper genome organization is essential for genome function and stability. Disruptions to this organization can lead to detrimental effects and the transformation of cells into diseased states. Individual chromosomes and their subregions can move or rearrange during transcriptional activation, in response to DNA damage, and during terminal differentiation. Techniques such as fluorescence in situ hybridization (FISH) and chromosome conformation capture (e.g., 3C and Hi-C) have provided valuable insights into genome architecture. However, these techniques require cell fixation, limiting studies of the temporal evolution of chromatin organization in detail. Our understanding of the heterogeneity and dynamics of chromatin organization at the single-cell level is still emerging. To address this, clustered regularly interspaced short palindromic repeats (CRISPR)/dead Cas9 (dCas9) systems have been repurposed for precise live-cell imaging of genome dynamics. This protocol uses a system called CRISPRainbow, a powerful tool that allows simultaneous targeting of up to seven genomic loci and tracks their locations over time using spectrally distinct fluorescent markers to study real-time chromatin organization. Multiple single-guide RNA (sgRNA), carrying specific RNA aptamers for labeling, can be cloned into a single vector to improve transfection efficiency in human cells. The precise targeting of CRISPRainbow offers distinct advantages over previous techniques while also complementing them by validating findings in live cells. Key features • Simultaneous imaging of up to seven specific genomic loci in living cells. • Multicolor imaging using a single CRISPR system from <i>Streptococcus pyogenes.</i> • Signal amplification through targeting repetitive sequences. • Targeting endogenous DNA without the need for foreign DNA insertion.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 17","pages":"e5432"},"PeriodicalIF":1.1,"publicationDate":"2025-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12423276/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145066750","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 Capture of Chromatin-Associated RNA and Global RNA-RNA Interactions With Reduced Input Requirements.","authors":"Cheng Ding, Guoting Chen, Shiping Luan, Yuanyuan Gong, Cuilin Gui, Chen Yang, Zihe Xiang, Junjie Du, Mohamed F Foda, Jiapei Yan, Xingwang Li","doi":"10.21769/BioProtoc.5430","DOIUrl":"10.21769/BioProtoc.5430","url":null,"abstract":"<p><p>Chromatin-associated RNAs (caRNAs) have been increasingly recognized as key regulators of gene expression and genome architecture. A few technologies, such as ChRD-PET and RedChIP, have emerged to assess protein-mediated RNA-chromatin interactions, but each has limitations. Here, we describe the TaDRIM-seq (targeted DNA-associated RNA and RNA-RNA interaction mapping by sequencing) technique, which combines Protein G (PG)-Tn5-targeted DNA tagmentation with in situ proximity ligation to simultaneously profile caRNAs across genomic regions and capture global RNA-RNA interactions within intact nuclei. This approach reduces the required cell input, shortens the experimental duration compared to existing protocols, and is applicable to both mammalian and plant systems. Key features • A multi-omics sequencing strategy. • Compatible with mammalian and plant systems. • Profiling of epigenome, RNA-DNA, and RNA-RNA interactomes.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 17","pages":"e5430"},"PeriodicalIF":1.1,"publicationDate":"2025-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12423280/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145066790","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":"New Approach to Detect and Isolate Rhamnogalacturonan-II in <i>Arabidopsis thaliana</i> Seed Mucilage.","authors":"Dayan Sanhueza, Susana Saez-Aguayo","doi":"10.21769/BioProtoc.5433","DOIUrl":"10.21769/BioProtoc.5433","url":null,"abstract":"<p><p>Rhamnogalacturonan-II (RG-II) is one of the least studied domains of pectin, primarily due to its low abundance, the lack of reliable antibodies, and the complexity of its structure. The present study builds upon existing protocols and procedures used to analyse RG-II in tissues where it is more abundant, combining and adapting them for the isolation of RG-II from <i>Arabidopsis</i> seed mucilage-a structure previously thought to lack RG-II. By applying these adapted methods, we first confirmed the presence of RG-II in seed mucilage and subsequently succeeded in isolating it from a tissue where it is typically present in low abundance, thereby enabling future studies on this previously overlooked component. Key features • An efficient RG-II isolation protocol offers the opportunity to simplify and deepen the study of RG-II. • It provides reliable yields. • The protocol allows analysis of RG-II synthesis or structural changes affecting dimerisation in mutant lines using <i>Arabidopsis</i> seed mucilage.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 17","pages":"e5433"},"PeriodicalIF":1.1,"publicationDate":"2025-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12423279/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145066671","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":"Quantitative Microscopy for Cell-Surface and Cell-Cell Interactions in Immunology.","authors":"Beatriz Díaz-Bello, Dalia El Arawi, Rémy Torro, Patrick Chames, Kheya Sengupta, Laurent Limozin","doi":"10.21769/BioProtoc.5427","DOIUrl":"10.21769/BioProtoc.5427","url":null,"abstract":"<p><p>Cell-surface and cell-cell interaction assays are fundamental for studying receptor-ligand interactions and characterizing cellular responses and functions. They play a critical role in diagnostics and in modulating immune system activity for therapeutic applications, notably in cancer immunotherapy. By providing time-lapsed and cell-level direct observation of the sample, optical microscopy offers strong advantages compared to current go-to techniques, which are typically either ensemble methods (e.g., measuring cell populations) or indirect readouts (e.g., impedance for adherent cells). This protocol describes two complementary microscopy-based assays: (1) a cell-surface ligand binding assay to quantify dynamic interactions between human primary Natural Killer (NK) cells and a cancer-mimicking surface, and (2) a cell-cell interaction assay to evaluate antibody-dependent cell cytotoxicity (ADCC) mediated by NK cells targeting tumor cells. Additionally, the protocol uses Celldetective, a new open graphical user interface for quantitative analysis of cell interaction dynamics from 2D time-lapse microscopy datasets. Although applied here to primary immune cells, these methods are adaptable to various cell types, including other immune cells, fibroblasts, and cancer cells. This approach enables direct observation and quantification of cellular morphology, motility, cell-cell interactions, and dynamic behaviors at single-cell resolution over time, facilitating detailed analysis of mechanisms such as cell death, migration, and immune synapse formation. Key features • End-to-end protocol for antibody evaluation by optical microscopy on living cells using simple reagents, followed by full open-source software image analysis and data rendering • Quantitative analysis of cell-surface interactions using label-free imaging to study the dynamic spreading of NK cells on antibody-coated surfaces under different antibody concentrations. • High-resolution evaluation of antibody-dependent cell cytotoxicity in NK-cancer cells co-culture using fluorescence imaging, deep learning-based death detection, and synchronized single-cell measurements.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 17","pages":"e5427"},"PeriodicalIF":1.1,"publicationDate":"2025-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12423278/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145066705","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}