Bio-protocol最新文献

{"title":"Uptake Assay of Ram Seminal Plasma Extracellular Vesicles to Sperm.","authors":"Tomas Armani, Anabella R Nicolli, Lucia Zalazar, Juan I Lobo, Monserrat Buendía Arellano, Federico A Hozbor, Sofia Rio, Silvina Perez Martinez, Andreina Cesari","doi":"10.21769/BioProtoc.5653","DOIUrl":"https://doi.org/10.21769/BioProtoc.5653","url":null,"abstract":"<p><p>Extracellular vesicles (EVs) are critical mediators of cell-cell communication and play a key role in male reproductive biology by modulating sperm function. This protocol describes a robust and reproducible workflow for isolating EVs from ram seminal plasma using size-exclusion chromatography (SEC) and assessing their uptake by ram spermatozoa. In contrast to ultracentrifugation-based methods, SEC provides a gentle and more efficient isolation approach that preserves EV integrity and functionality. A central innovation of this protocol is the use of carboxyfluorescein succinimidyl ester (CFSE)-labeled seminal plasma EVs (SP-EVs) to evaluate their incorporation into sperm cells through two complementary detection platforms: (i) flow cytometry with standard resolution and (ii) confocal microscopy, for spatial confirmation of EV-sperm interactions. By bridging the gap between EV isolation and functional analysis, this protocol provides a valuable tool for investigating the role of EV-cell interactions. Specifically, it offers potential applications in male fertility preservation, biomarker discovery, and the development of EV-based therapeutic strategies in reproductive medicine. Key features • Provides a gentle, SEC-based EV isolation method optimized for ram seminal plasma, suitable for preserving vesicle integrity in studies of male reproductive biology. • Integrates EV purification with functional assays, enabling direct evaluation of EV-sperm interactions through confocal microscopy and flow cytometry. • Includes a reproducible CFSE-labeling strategy tailored for seminal plasma EVs, ensuring consistent detection of vesicle uptake by ram spermatozoa. • Designed for applications in fertility research, offering a workflow compatible with biomarker discovery, cryopreservation studies, and development of EV-based reproductive interventions.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 7","pages":"e5653"},"PeriodicalIF":1.1,"publicationDate":"2026-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13067157/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147679397","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":"MDISCO: A High-Throughput Tissue-Clearing Protocol for Preservation of Endogenous Fluorescence in Whole Mouse Brains.","authors":"Madeline Martinez, Jake Thornberry, Akihiko Ozawa, Lawrence Toll","doi":"10.21769/BioProtoc.5649","DOIUrl":"https://doi.org/10.21769/BioProtoc.5649","url":null,"abstract":"<p><p>Organic solvent-based tissue clearing methods are widely used for whole-brain imaging but often compromise endogenous fluorescence. Existing protocols, such as iDISCO and fluorescence-preserving variants, have improved optical transparency but still present trade-offs between fluorescence retention, tissue stability, and workflow complexity. Here, we present MDISCO, a modified iDISCO-based clearing protocol designed to enhance preservation of endogenous fluorescence while maintaining high transparency and stable tissue morphology. MDISCO is directly compared with FDISCO+, an established fluorescence-preserving protocol, for the preservation of endogenous tdTomato and YFP. Performance across clearing steps is evaluated by measuring brain weight, anteroposterior and mediolateral dimensions, and optical transparency before and after solvent clearing and refractive index matching. Fluorescence preservation is assessed using whole-brain light-sheet microscopy with standardized imaging parameters to enable direct comparison. This protocol provides an accessible and high-throughput, reproducible workflow for solvent-based clearing with robust endogenous fluorescence preservation, offering clear advantages for whole-brain 3D imaging of genetically encoded fluorescent reporters. Key features • Preserves endogenous tdTomato and YFP fluorescence in whole mouse brains without signal amplification through immunolabeling. • Improves optical clarity and cellular resolvability while maintaining anatomical integrity. • Supports high-throughput \"clearing\" of whole-tissue samples.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 7","pages":"e5649"},"PeriodicalIF":1.1,"publicationDate":"2026-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13067151/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147679414","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":"Microinjection of Synthetic Peptides Into <i>Caenorhabditis elegans</i>.","authors":"Hayao Ohno, Takanori Ida, Yuichi Iino","doi":"10.21769/BioProtoc.5640","DOIUrl":"https://doi.org/10.21769/BioProtoc.5640","url":null,"abstract":"<p><p>The genome of the nematode <i>Caenorhabditis elegans</i> encodes at least 160 predicted peptide precursor genes that can generate over 300 bioactive peptides, the functions of most of which remain unknown. Phenotypes resulting from deletion or transgenic expression of peptide genes are readily assayed, but genetic dissection of individual peptide activities is often confounded when a single gene encodes multiple peptides or when distinct peptides act redundantly. Here, we describe a protocol for direct microinjection of chemically synthesized peptides into individual worms. This approach permits investigation of the effects of an individual peptide while providing precise temporal control over peptide delivery. Key features • Direct injection of chemically synthesized peptides into nematodes. • In vivo effects can be analyzed within one day after peptide delivery. • Full control over peptides, chemical modifications, genetic background, and timing of delivery. • The approach may be adaptable to non-peptide compounds.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 7","pages":"e5640"},"PeriodicalIF":1.1,"publicationDate":"2026-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13067155/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147679422","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 Method for Estimating the Spatiotemporal Distribution of Phenoloxidase Proteins in Insect Tissues.","authors":"Yusuke Nakatani, Yuji Matsuoka, Shinichi Morita, Teruyuki Niimi","doi":"10.21769/BioProtoc.5646","DOIUrl":"https://doi.org/10.21769/BioProtoc.5646","url":null,"abstract":"<p><p>Laccase2 (Lac2), a member of the phenoloxidase (PO) family, is an essential oxidase for melanin pigmentation in insects. The identification of the in vivo spatial distribution of Lac2 is crucial for understanding the molecular mechanisms underlying color pattern formation. However, it is technically difficult to determine the distribution because Lac2 expression peaks at late pupal stages, when adult cuticle sclerotization has already begun. Here, we report a simple and rapid protocol for estimating the distribution of endogenous PO proteins, prophenoloxidases (proPOs) and phenoloxidases (POs), in insect tissues. In this method, the spatial distribution of endogenous PO proteins is estimated based on staining patterns formed by dopamine melanin synthesis in tissues incubated in a solution containing isopropanol and dopamine. We validated that tissues collected at approximately 80% of the total pupal duration yielded staining patterns corresponding to adult melanin-forming regions in three insect species. By comparing staining patterns across developmental stages, this protocol enables estimation of the timing of color pattern formation. Furthermore, the contrast between stained and unstained regions within the same tissue allows region-specific sampling, thereby facilitating an investigation of the underlying molecular mechanisms regulating spatial PO distribution. Taken together, this method facilitates the study of melanin biosynthesis and enables the identification of the genes involved in regulating color pattern formation. This protocol does not require antibodies, transgenic lines, or specialized equipment and can be completed within a short time frame. Its effectiveness has been validated in multiple coleopteran and lepidopteran species, demonstrating its broad applicability as a versatile tool for studying insect pigmentation and color pattern formation. Key features • A simple tissue staining protocol to estimate the spatial distribution of endogenous PO proteins without the use of antibodies or transgenic lines. • Comparing staining patterns across different developmental stages to estimate both the spatial distribution of PO proteins within tissues and the timing of color pattern formation. • RNAs can be extracted from the tissue after staining, enabling gene expression analyses between stained and unstained regions. • This protocol has been validated in two coleopteran species and one lepidopteran species, demonstrating broad applicability across diverse insect taxa.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 7","pages":"e5646"},"PeriodicalIF":1.1,"publicationDate":"2026-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13067149/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147679393","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":"Recovery and Expansion of Patient-Derived Glioblastoma Cells After Long-term Cryopreservation.","authors":"Wannawat Khotchawan, Chanchao Lorthongpanich, Pakpoom Kheolamai, Sith Sathornsumetee, Surapol Issaragrisil","doi":"10.21769/BioProtoc.5661","DOIUrl":"https://doi.org/10.21769/BioProtoc.5661","url":null,"abstract":"<p><p>Patient-derived glioblastoma (GBM) cells are valuable models for GBM research due to their rarity and the highly lethal nature of this cancer. Preserving these cells through long-term cryopreservation is therefore essential for advancing future investigations. However, recent studies have reported that standard cell recovery protocols are inefficient, resulting in poor cell survival and limited regrowth. Here, we established an optimized culture protocol that enhances the recovery and expansion of patient-derived GBM cells by combining Matrigel with an increased concentration of fetal bovine serum (FBS). This approach significantly improves cell attachment and recovery after thawing cells that have been cryopreserved for more than a decade. Importantly, the recovered cells retain key phenotypic characteristics and remain suitable for downstream applications, including drug testing and spheroid formation. Together, this optimized protocol provides a novel strategy to increase the availability of patient-derived GBM cells by improving their efficient recovery from long-term cryopreservation, thereby maximizing their utility in GBM research. Key features • Optimized for recovery of low-viability adherent cells, including long-term cryopreserved patient-derived GBM. • Combined use of Matrigel coating and elevated FBS to enhance post-thaw attachment and recovery. • Recovered cells maintain their morphology, marker expression, and functionality. • Simple, effective protocol applicable to GBM and potentially to other adherent cell types.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 7","pages":"e5661"},"PeriodicalIF":1.1,"publicationDate":"2026-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13067158/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147679453","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":"Preparing Adjuvanted Nanoliposomes for Applications Toward Recombinant Influenza Vaccine Development.","authors":"Zachary R Sia, Wei-Chiao Huang, Matthew Willadsen, Hilliard L Kutscher, Jonathan F Lovell, Bruce A Davidson","doi":"10.21769/BioProtoc.5662","DOIUrl":"https://doi.org/10.21769/BioProtoc.5662","url":null,"abstract":"<p><p>Nanoparticle vaccines can provide advantages over traditional vaccine methodologies, including adjuvant delivery to enhance the effectiveness of recombinant antigens. Many approaches exist to formulate different vaccine nanoparticles, which are designed for different biomolecular cargos, adjuvant compositions, and disease targets. Here, a protocol is described to produce nanoliposomes whose surface is decorated with recombinant protein influenza antigens with monophosphoryl lipid A and QS-21 adjuvants incorporated into the lipid bilayer for protection against influenza infection. This protocol includes methods for producing adjuvanted liposomes and coupling with His-tagged antigens for surface decoration of the particle. This allows for a rapid methodology of producing immunogenic antigen-presenting liposomes that can be tailored to display a combination of influenza surface antigens. Key features • This protocol uses recombinant proteins, which can be adapted to the desired strain sequence. • Production of this influenza vaccine formulation uses in vitro techniques, eliminating the need for virus growth in eggs. • Additional lipid adjuvants can be included in the liposome production step to be incorporated into the surface membrane. • Cobalt-porphyrin phospholipid provides a flexible platform for binding His-tag-bearing proteins.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 7","pages":"e5662"},"PeriodicalIF":1.1,"publicationDate":"2026-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13067150/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147679369","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":"Fluorescence-Based Absent Allele-Specific Amplification (FAASA) for High-Throughput Detection of Absent Alleles.","authors":"Katherine L D Running, Sudeshi Seneviratne, Zengcui Zhang, Gurminder Singh, Jason D Fiedler, Justin D Faris","doi":"10.21769/BioProtoc.5633","DOIUrl":"https://doi.org/10.21769/BioProtoc.5633","url":null,"abstract":"<p><p>In wheat and other crops, some genes display presence/absence variation, and it is occasionally beneficial to select for the absent allele to remove a functional gene. However, current high-throughput genotyping methods used to detect the absence of genes tend to be inconsistent and inconclusive. Kompetitive allele-specific PCR (KASP) and PCR allele competitive extension (PACE) are two well-established methods for allele-specific polymerase chain reaction (AS-PCR) assays, each using fluorescence resonance energy transfer (FRET) to generate a signal for each allele, typically targeting biallelic single-nucleotide polymorphisms. KASP and PACE methods are more difficult to apply to alleles with presence/absence variation because the lack of amplification of the absent allele is indistinguishable from a failed PCR. Here, we present a multiplex fluorescence-based absent allele-specific amplification (FAASA) method using the PACE marker system (compatible with KASP markers) to detect the absence of one particular or all alleles of a target sequence using a primer mix consisting of one target-specific primer pair (TSP) and a second primer set specific to a highly conserved endogenous gene known as a core gene-specific primer pair (CGSP). The forward primer of each pair is tagged with a 5' terminal tail complementary to dye-labeled oligonucleotides in commercially available FRET cassettes. Lines that amplify only the core gene do not carry the target, while lines that amplify both the core gene and the target carry alleles of both the core gene and the target. The inclusion of the CGSPs allows researchers to confidently distinguish lines with absent alleles of the target from lines with failed PCR reactions, which can happen due to various reasons, including inadequate DNA quality or quantity. Key features • A robust, affordable, high-throughput genotyping method for genes or other target sequences with presence/absence variation. • FAASA markers can be easily incorporated into established marker-assisted selection programs in labs using KASP and/or PACE markers. • FAASA markers can also be used for other genotyping applications like GWAS, QTL, or bi-parental mapping studies. • Easily adaptable to different targets and species of interest.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 6","pages":"e5633"},"PeriodicalIF":1.1,"publicationDate":"2026-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13037777/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147596724","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":"Tandem RNA and Protein Extraction: A Platform for Maximizing the Use of Limited Ex Vivo Tissue Samples.","authors":"Ciarán Kennedy, Braden Millar, Luke J Conroy, Mariam Marai, Mary Barry, Martin O'Donohoe, Orina Belton, Eoin Brennan, Catherine Godson, Monica de Gaetano","doi":"10.21769/BioProtoc.5643","DOIUrl":"https://doi.org/10.21769/BioProtoc.5643","url":null,"abstract":"<p><p>Human tissue samples represent the gold standard for obtaining clinically relevant and translatable insight into disease processes that in vitro systems cannot fully reproduce. However, patient-derived samples are often limited in size and availability, limiting the number of downstream assays that can be performed. To maximize the use of invaluable human samples, we present a protocol for the tandem extraction of high-quality RNA and protein from the same tissue section. This method has been optimized for 15-30 mg tissue sections, enabling more experimental conditions and technical replicates, while minimizing intrasample variability associated with heterogeneous tissues. This protocol also avoids potentially hazardous solvents present in phenol-chloroform-based methods such as TRIzol, providing a safer and more accessible workflow without compromising biomolecule integrity. This protocol was developed and validated using atherosclerotic plaque tissue from carotid endarterectomy, a very challenging tissue type to work with due to extensive calcification, necrosis, and limited surgical availability. We have also validated this method using mouse aortic tissue and cultured THP-1 cells, demonstrating its versatility across sample input types. As this protocol relies on standard column-based RNA extraction kits and commonly available reagents for protein precipitation and extraction, this methodology is widely accessible and easy to implement as a standard, streamlined workflow. Key features • Advances ex vivo models of human disease (e.g., atherosclerosis) by maximizing the use of patient-derived tissue samples. • Minimizes patient variability by generating directly comparable RNA and protein readouts. • Minimizes the number of patient samples required and maximizes the output from each sample. • Stabilizes RNA integrity from tissue without impacting the quality of proteins derived from the same sample.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 6","pages":"e5643"},"PeriodicalIF":1.1,"publicationDate":"2026-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13037783/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147596707","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 Guide to Reproducible Cellulose Synthase Density and Speed Measurements in <i>Arabidopsis thaliana</i>.","authors":"Jan Y Xue, Shawn D Mansfield, Lacey A Samuels, Arun Sampathkumar, René Schneider","doi":"10.21769/BioProtoc.5634","DOIUrl":"https://doi.org/10.21769/BioProtoc.5634","url":null,"abstract":"<p><p>Cellulose synthase complexes (CSCs) play a central role in plant cell wall formation. Their dynamic behavior at the plasma membrane leads to the deposition of cellulose microfibrils into the apoplastic space, thereby shaping the architecture and mechanical properties of the cell wall. Although previous imaging studies have provided important insights into CSC dynamics and localization, standardized and reproducible workflows for quantitative measurements of CSC speed and density remain limited. Here, we present a reproducible live-cell imaging and analysis workflow for quantifying the speed and density of fluorescently labeled CSCs at the plasma membrane in <i>Arabidopsis thaliana</i>. The protocol integrates optimized spinning-disk confocal imaging, surface-based projection of z-stack recordings, automated detection of diffraction-limited CSCs foci, and kymograph-based speed measurements using freely available tools in Fiji. While selected steps, such as region of interest definition and parameter selection for spot detection or trajectory analysis, remain user-guided, these decisions are constrained to well-defined stages within an otherwise standardized pipeline, thereby reducing variability and improving reproducibility across experiments. The workflow has been validated across multiple tissues, reporter lines, genetic backgrounds, and perturbation conditions in <i>Arabidopsis</i> and enables robust comparative analysis of CSC dynamics. Beyond CSCs, this workflow is expected to be adaptable to other fluorescently labeled proteins that appear as diffraction-limited foci at or near the plasma membrane. Key features • Enables accurate CSC speed and density measurements during both primary and secondary cell wall formation using spinning-disk confocal time-lapse imaging. • Combines surface-projection, kymograph analysis, and high-throughput particle detection to quantify CSC dynamics even in crowded or low-signal plasma membrane regions. • Provides a standardized analysis workflow validated across multiple <i>Arabidopsis</i> genotypes, including inducible systems and mutant backgrounds that possess altered cell wall biosynthesis. • Applicable to any fluorescently labeled diffraction-limited foci at or near the plasma membrane, extending the workflow beyond CSCs.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 6","pages":"e5634"},"PeriodicalIF":1.1,"publicationDate":"2026-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13037776/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147596715","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 Cost-Effective Method for Generating Spheroids From Triple-Negative Breast Cancer Cell Line (MDA-MB-231).","authors":"Ramón Cervantes-Rivera, Luisa Nirvana González-Fernández, Atalia Ziret Romero Rosas, Sandra Jetsamari Figueroa Ortíz, Alejandra Ochoa-Zarzosa, Joel E López-Meza","doi":"10.21769/BioProtoc.5641","DOIUrl":"https://doi.org/10.21769/BioProtoc.5641","url":null,"abstract":"<p><p>Breast cancer (BC) is the most frequently diagnosed malignancy in women and a leading cause of cancer-related mortality worldwide. Current clinical management relies on molecular classification-based on estrogen receptor (ER), progesterone receptor (PR), HER2, and Ki67 expression-to guide prognosis and therapy. Triple-negative breast cancer (TNBC), which lacks ER, PR, and HER2 expression, represents 15%-20% of cases and is characterized by aggressive behavior, early recurrence, and a paucity of targeted treatment options. These challenges underscore the urgent need for improved preclinical models that better recapitulate tumor biology to accelerate therapeutic discovery. While conventional monolayer (2D) cultures have contributed significantly to cancer research, they fail to mimic critical features of the three-dimensional (3D) tumor microenvironment (TME), thereby limiting clinical translation. To address this gap, 3D spheroid models have emerged as a powerful intermediary, more accurately replicating in vivo conditions such as cell-cell and cell-matrix interactions, nutrient and oxygen gradients, and the development of hypoxic cores. These features make spheroids a physiologically relevant platform for studying complex processes like metastasis, drug resistance, and treatment response. Here, we present a robust, simple, and cost-effective protocol for generating uniform 3D spheroids. Our method enables consistent monitoring of spheroid formation and growth over time, with quantitative, image-based size analysis to ensure reproducibility and scalability. Designed for flexibility, the protocol is broadly applicable across diverse cell types, effectively bridging the gap between traditional 2D cultures and complex in vivo studies. By providing an accessible and reliable model of the 3D TME, this protocol opens new avenues for high-throughput drug screening, mechanistic studies of tumor progression, and the advancement of personalized medicine strategies in breast cancer and beyond. Key features • Employs a cost-effective, lab-made agarose coating to create non-adherent surfaces in standard 96-well plates. • Optimized for reproducible spheroid formation from the aggressive MDA-MB-231 triple-negative breast cancer cell line. • Requires a brief orbital shaking step to promote homogeneous cell aggregation and formation of a single central spheroid per well. • Generates measurable spheroids within 96 h using only basic cell culture equipment and an orbital shaker. • Provides a clear workflow from spheroid formation to quantitative size analysis, using freely available (ImageJ) and widely used (GraphPad Prism) software.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 6","pages":"e5641"},"PeriodicalIF":1.1,"publicationDate":"2026-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13037779/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147596734","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}