Methods and Protocols最新文献

{"title":"Selection and Characterization of Cell Line-Virus Pairs for Sensitive Viral Detection Assays in Biopharmaceutical Testing.","authors":"Agnieszka Staniszewska, Agnieszka Piastowska-Ciesielska","doi":"10.3390/mps9040107","DOIUrl":"10.3390/mps9040107","url":null,"abstract":"<p><p>Ensuring viral safety is a critical aspect of biopharmaceutical production, requiring sensitive and reliable methods for detecting adventitious agents. In this study, we systematically evaluated the performance of selected cell line-virus combinations to identify optimal models for in vitro viral detection assays. Three cell lines (Vero, MRC-5 and BHK-21 [C-13]) and representative model viruses (Reovirus type 3, Adenovirus type 5, Human parainfluenza virus type 3, and Herpes simplex virus) were analyzed in terms of cytopathic effect (CPE) kinetics, morphology, and detection sensitivity. All tested systems demonstrated high analytical sensitivity, with limits of quantification (LOQ) reaching 0.01 TCID₅₀/mL for selected viruses. However, substantial differences were observed in infection dynamics and CPE morphology depending on the cell line-virus combination. BHK-21 [C-13] cells exhibited the most rapid and pronounced CPE for Reovirus type 3, enabling early and unambiguous detection. Vero cells provided robust and reproducible detection of Adenovirus type 5, characterized by well-defined cytopathic progression. MRC-5 cells showed controlled and consistent infection kinetics for both Human parainfluenza virus type 3 and Herpes simplex virus, allowing improved temporal resolution and interpretability. These findings demonstrate that assay performance depends not only on sensitivity but also on the kinetics and morphology of infection. Based on combined evaluation criteria, the following optimal cell line-virus pairs were identified: BHK-21 [C-13]/Reovirus type 3, Vero/Adenovirus type 5, and MRC-5/Human parainfluenza virus type 3 and Herpes simplex virus. The proposed approach supports rational selection of detection models and provides a preliminary descriptive framework for the development of routine visual screening assays in biopharmaceutical quality control.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13398311/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148579343","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":"Simplified and Rapid Preparation Protocol for Producing <i>Aloe Vera</i>-Based Natural Coagulant for Water Treatment.","authors":"Danieli Soares de Oliveira, Clainer Bravin Donadel","doi":"10.3390/mps9040106","DOIUrl":"10.3390/mps9040106","url":null,"abstract":"<p><p>Natural coagulants have emerged as potential alternatives to synthetic chemicals in water treatment, especially for decentralized and low-resource applications. However, many previously reported <i>Aloe vera</i>-based coagulant preparation methods rely on drying, powder production, distilled water extraction, refrigeration, or other laboratory-dependent procedures that increase operational complexity and limit practical implementation. This study presents a simplified and rapid protocol for producing an <i>Aloe vera</i>-based natural coagulant using accessible materials and simplified preparation steps. The proposed methodology consists of extracting <i>Aloe vera</i> g13el, homogenizing 2 g of fresh gel with 50 mL of tap water using a household blender, and applying simple paper filtration to obtain the liquid coagulant. The protocol can be completed in less than 10 min without specialized laboratory infrastructure, energy-intensive processing, or laboratory-grade reagents. Coagulation performance was evaluated using synthetic turbid water with initial turbidity levels of 100, 200, and 300 NTU. Significant turbidity reduction was observed under all tested conditions, with several samples reaching residual turbidity values close to or equal to 0 NTU after 50-60 min of sedimentation. The results demonstrate the potential of the proposed protocol as a rapid, reproducible, and accessible approach for future investigation in point-of-use and decentralized water treatment applications.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13397813/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148579396","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An Improved Method for the Isolation of Extrachromosomal DNA from the Pathogenic Free-Living Amoeba <i>Naegleria fowleri</i>.","authors":"Colm P Roster, James C Morris","doi":"10.3390/mps9040105","DOIUrl":"10.3390/mps9040105","url":null,"abstract":"<p><p>The pathogenic free-living amoeba <i>Naegleria fowleri</i> is the cause of primary amebic meningoencephalitis (PAM), a central nervous system infection that is almost always lethal. One of the unusual features of the amoebae is the presence of ~4000 copies of a nucleolar-localized closed circular extrachromosomal ribosomal DNA element (CERE) that encodes the cell's ribosomal RNA repertoire. It has historically been challenging to purify large quantities of CERE, limiting our understanding of the nucleic acid. Here, we describe a methodology for CERE purification that improves yield, reduces processing times, and maintains the integrity of the plasmid. This approach will enable the study of this unique DNA architecture, advancing our understanding of the pathobiology of the organism.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13398248/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148579416","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":"Non-Healing Wound Model in Diabetic C57BL/6 Mice.","authors":"Lyubov A Rzhanova, Ekaterina V Kuzmenko, Alena A Permyakova, Andrei A Riabinin, Evgenii S Ruchko, Maria B Chernysheva, Ekaterina A Vorotelyak, Elena I Morgun","doi":"10.3390/mps9040104","DOIUrl":"10.3390/mps9040104","url":null,"abstract":"<p><p><b>Background</b>: This study focuses on developing a model of a non-healing wound that recapitulates the pathogenesis of the corresponding human pathology. <b>Methods</b>: A non-healing wound was modeled in mice with streptozotocin-induced diabetes. The following parameters were assessed: re-epithelialization, epidermal hypertrophy, wound contraction, relief index, angiogenesis, and granulation tissue maturation. These parameters were compared between diabetic mice and healthy controls. <b>Results</b>: The proposed model demonstrated a significant delay in regenerative processes compared to healthy animals. <b>Conclusions</b>: These findings <b>support the relevance</b> of this model to human pathology and <b>indicate that it may be applicable</b> for preclinical studies of drugs aimed at promoting wound regeneration.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13398050/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148579385","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":"Standardized Protocol for Comprehensive, Non-Invasive Phenotyping of Atrial Myopathy in Sprague-Dawley Rat Models of Metabolic Syndrome Using Clinical-Grade Echocardiography and Electrophysiology Systems.","authors":"Ardian Rizal, Mohammad Saifur Rohman, Fatchiyah Fatchiyah, Hidayat Sujuti, Anna Fuji Rahimah, Wella Karolina, Victor Alvianoes Guterez Hose, Mokhammad Afifudin","doi":"10.3390/mps9040103","DOIUrl":"10.3390/mps9040103","url":null,"abstract":"<p><p><b>Background:</b> Small animal models are essential for atrial fibrillation (AF) research. Researchers in AF use an electrocardiogram (ECG), echocardiography and invasive electrophysiology study (EPS) to assess atrial structural and electrical remodeling. In relatively smaller cardiac structures and rapid heart rates, the examination can be challenging without special tools designed for animal study. Moreover, conventional invasive EPSs often cause significant trauma, alter autonomic tone, and limit longitudinal evaluations. This study aimed to evaluate the feasibility of repurposing hospital-grade medical devices for the non-invasive, multi-modality assessment of atrial myopathy in a rat model of metabolic syndrome (MetS). <b>Methods:</b> A total of 12 male Sprague-Dawley rats underwent the multi-modality assessment. Structural remodeling was evaluated using hospital-grade echocardiography (8-12 MHz) to measure left atrial (LA) dimensions and volume. Surface ECG was used to determine P-wave duration. Electrical remodeling and AF inducibility were assessed using transesophageal pacing (TEP)-based EPS, evaluating the atrial effective refractory period (AERP), sinus node recovery time (SNRT), and response to rapid atrial burst pacing. <b>Results:</b> The protocols showed high procedural safety (survival rate 91.67%) and successfully characterized atrial myopathy. Surface ECG showed marked intra-atrial conduction delay with prolonged P-wave duration in the MetS group (30.17 ± 4.62 vs. 22.33 ± 1.86 ms, <i>p</i> < 0.05). Echocardiography revealed signs of structural remodeling in the MetS group, evidenced by marked prolonged Isovolumic Relaxation Time (IVRT: 35.602 ± 3.043 vs. 19.187 ± 3.631 ms; <i>p</i> < 0.001) and increased Left Atrial Area (0.223 ± 0.0556 vs. 0.134 ± 0.033; <i>p</i> = 0.007). Furthermore, TEP-based EPS quantified electrical remodeling. The MetS group had shorter AERP (73.33 ± 10.33 ms vs. 120.00 ± 34.06 ms; <i>p</i> = 0.010) and Corrected SNRT (100.67 ± 53.98 ms) versus controls (208.33 ± 76.97 ms; <i>p</i> = 0.018). The MetS group exhibited a higher absolute AF inducibility rate (50%, three out of six rats) compared to the SH group (33.3%, two out of six rats). <b>Conclusions:</b> The integration of surface ECG, echocardiography, and TEP-based EPS provides a safe, highly reproducible, and comprehensive method for evaluating both structural and electrical components of atrial myopathy in small animal models, allowing for robust longitudinal studies.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13398038/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148579434","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":"Evaluating Effectiveness of the FiTeens Intervention for Health Behavior Change in Students: A Study Protocol.","authors":"Taavi Rand, Henri Tilga, Ángel Abós, Luis García-González, Sergio Diloy Peña, Rafael Burgueño-Mengibar, Andre Koka","doi":"10.3390/mps9040101","DOIUrl":"10.3390/mps9040101","url":null,"abstract":"<p><p>(1) Background: Previous school-based interventions have addressed adolescent health behaviors such as physical activity, screen time, and sleep, but have predominantly targeted these behaviors independently rather than simultaneously. The Erasmus+ project FiTeens developed an integrated intervention combining theoretical content, videos, infographics, and interactive tasks to promote multiple health behaviors concurrently. The objective of the current article is to present the protocol for a school-based intervention study designed to examine the effects of the FiTeens program on adolescents' physical activity, screen time, and sleep behaviors. We hypothesize that students receiving the FiTeens intervention will demonstrate increased physical activity, reduced screen time, and improved sleep outcomes compared with students in the control group. (2) Methods: Teachers will be introduced to the FiTeens tools prior to delivering the intervention to students in grades 5-9. Students will participate in an eight-week intervention program combining structured lessons and behavior-change challenges. Primary outcomes include changes in physical activity, screen time, and sleep duration and quality. Secondary outcomes include psychological determinants such as motivation and behavioral intentions. Data will be collected at baseline and at 1-, 3-, and 6-month follow-ups and analyzed using repeated measures ANOVA. (3) Expected results: The study will evaluate whether the intervention may contribute to improvements in health-related behaviors among adolescents, including increased physical activity, reduced screen time, and improved sleep outcomes. (4) Conclusions: The intervention based on FiTeens tools could have the potential to promote healthier lifestyle behaviors among students by increasing physical activity during leisure time, supporting the effective limitation of screen time and enhancing bedtime routines to improve sleep quality.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13397910/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148579421","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 Miniaturised Protocol for Feeding Measurements in Daphnids.","authors":"Izabela Antepowicz, Antonia Despotidi, Emma Rowan, Mbuyiselwa Shadrack Moloi, Silke Aulhorn, Harry Esmonde, Konstantinos Grintzalis, Eberhard Küster","doi":"10.3390/mps9040102","DOIUrl":"10.3390/mps9040102","url":null,"abstract":"<p><p>Daphnids, commonly known as water fleas, are freshwater planktonic microcrustacean species used as model organisms in ecotoxicology, particularly in regulatory frameworks that adhere to OECD and ISO standards. Mortality is the most common endpoint in toxicity testing; however, more sensitive indicators are required to assess sublethal acute effects of pollutants. The use of feeding impairment as a toxicity phenotypic endpoint in daphnids is considered a cost-effective approach that aligns with the 3Rs principle (Replace, Reduce, Refine) and is more physiologically and environmentally relevant. Current feeding methods are inefficient due to the large test volumes and extended incubation periods required. In this paper, we present a miniaturised protocol to assess feeding behaviour following exposure to chemicals in daphnids. The method is based on the consumption of algae, which is measured with chlorophyll fluorescence. The optimised protocol is more robust and rapid, and results can be obtained in 30 min and in a 96-well plate. Responses in feeding rate were investigated using this miniaturised protocol following exposure to a range of prevalent pollutants, which include two metals and, as a more realistic sample, a leachate from smoked cigarette filters. All three pollutants were tested at sublethal concentrations. This method provides an efficient approach to assess the toxicity of chemicals and water quality.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13398296/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148579387","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An Efficient TetR/TetO-Integrated Packaging System for Fowl Adenovirus 4 Vector Carrying Toxic Transgene.","authors":"Qian-Wen Ma, Zhi Li, Zhi-Chao Zhang, Xiao-Juan Guo, Xiao-Hui Zou, Tao Hung, Zhuo-Zhuang Lu","doi":"10.3390/mps9030100","DOIUrl":"10.3390/mps9030100","url":null,"abstract":"<p><p>Adenoviral vectors are widely used for gene therapy and vaccine development. To circumvent pre-existing immunity against commonly used human adenovirus type 5, vectors based on rare human serotype or animal adenoviruses have attracted increasing interest. Previously, we constructed vectors based on fowl adenovirus 4 (FAdV-4) and replaced the knob of FAdV-4 fiber2 with that of FAdV-1 fiber1 to generate FAdV4-CF1K vectors with enhanced transduction efficiency in human cells. In this study, we aimed to modify the packaging system to efficiently produce FAdV-4 vectors carrying transgenes toxic to viral replication. Chicken LMH cells failed to form colonies at low seeding densities. We collected used medium from LMH cell cultures and used it as a supplement to adapt LMH cells, generating the colony-competent subclone LMH-C3532. A lentiviral vector encoding a codon-optimized tetracycline repressor (tetR) was transduced into LMH-C3532 to establish a tetR-integrated cell line, LMH-tetR24. An adenoviral plasmid, pKFAV4-CF1K-CtG, was constructed in which a tetracycline operator (tetO)-bearing CMV promoter controlled GFP expression. The SwaI-flanked GFP in this plasmid was replaced with the HA gene from an H5N1 influenza virus to generate pKFAV4-CF1K-CtHA. Linearized adenoviral plasmids were transfected into LMH-tetR24 cells, and recombinant FAdV4-CF1K-CtG and FAdV4-CF1K-CtHA viruses were successfully rescued, amplified, and purified. When infected with FAdV4-CF1K-CtG at various multiplicities of infection (MOI), the progeny virus yield from LMH-tetR24 cells was 4-10 times higher than that from LMH-C3532 cells. For FAdV4-CF1K-CtHA, the yield difference between the two cell lines was even more pronounced, reaching 3-4 orders of magnitude. Overexpression of HA in LMH-C3532 cells negatively affected FAdV4-CF1K-CtHA replication, resulting in smaller and fewer plaques. In conclusion, by separately integrating tetR into packaging cells and TetO into the adenoviral plasmid, we established a system that can be routinely used to package FAdV-4 vectors. Notably, this system facilitates the propagation of FAdV-4 vectors carrying toxic transgenes.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 3","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-06-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13306192/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148318069","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":"Exploring Barriers and Facilitators to COVID-19 Vaccination Uptake Among Individuals with Mental Illness in the Australian Healthcare System: A Qualitative Study Protocol.","authors":"Soumitra Das, James Killian, Mahesh Jayaram, Naveen Thomas, Chi Jonasi","doi":"10.3390/mps9030099","DOIUrl":"10.3390/mps9030099","url":null,"abstract":"<p><p>Individuals living with mental illness face disproportionately higher COVID-19 morbidity and mortality than the general population. Despite their prioritisation in Australia's national vaccination rollout, vaccination rates among this population are significantly lower than those without mental illness. No previous study has employed a qualitative research paradigm to explore the barriers and facilitators to COVID-19 vaccination among people with mental illness in the Australian context. This qualitative study will employ Braun and Clarke's reflexive thematic analysis framework. Participants will be recruited through Western Health Mental Health and Wellbeing Services in Victoria, Australia. Semi-structured individual interviews will be conducted with approximately 17-20 participants (aged 18-65) who have a DSM-5 diagnosed mental illness and any experience (vaccinated or unvaccinated) with COVID-19 vaccination. Interviews will be audio-recorded, transcribed, and then analysed and collated into themes using NVivo software. Code saturation will guide the final sample size. This study aims to produce a rich thematic map that captures the experience of individual, illness, and system-level barriers and facilitators to COVID-19 vaccination in this cohort. Findings are anticipated to inform targeted public health interventions to improve equitable vaccine uptake and contribute to closing the mortality gap for those with mental illness. This protocol has been approved by the Western Health Low Risk Ethics Panel (ERM ID: 113351).</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 3","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-06-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13305169/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148318176","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":"In Vitro Capacitation in Boar Sperm: Evaluation of Selected Detection Techniques.","authors":"Barbora Klusackova, Zuzana Pilsova, Katerina Nemeckova, Aneta Pilsova, Pavla Postlerova","doi":"10.3390/mps9030098","DOIUrl":"10.3390/mps9030098","url":null,"abstract":"<p><p>Sperm capacitation is essential for fertilization and involves coordinated changes in membrane organization, ion fluxes, and intracellular signaling. However, commonly used detection methods may reflect different biological events, which can be strongly influenced by experimental methodology. This study critically evaluated fluorescence-based approaches for assessing capacitation in boar spermatozoa, focusing on their specificity, interpretative limits, and methodological sensitivity. Ejaculated boar spermatozoa were incubated under in vitro capacitating conditions in TALP medium. Selected samples were subsequently treated with calcium ionophore to induce the acrosome reaction (AR). Phosphotyrosine (PTyr) immunofluorescence was assessed using five fixation and labeling protocols, acrosin redistribution was evaluated with the ACR.2 antibody, calcium ion redistribution was assessed using chlortetracycline (CTC) fluorescence, and acrosomal responsiveness was monitored by peanut agglutinin (PNA) lectin labeling. PTyr immunofluorescence was highly dependent on fixation protocol, indicating marked methodological sensitivity. Acrosin immunodetection revealed a clear capacitation-associated redistribution from weak or diffuse staining to a well-defined acrosomal pattern, whereas ionophore treatment caused a pronounced signal loss consistent with acrosomal exocytosis. PNA labeling confirmed that capacitation alone did not increase spontaneous acrosome loss, whereas ionophore treatment induced a robust AR. CTC staining showed a significant shift from whole-head pattern to acrosome in TALP-treated spermatozoa, indicating capacitation-associated Ca²⁺ redistribution. Together with CTC and Western blot data, these findings show that sperm capacitation status should be evaluated using multiple complementary markers rather than a single gold-standard assay.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 3","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13304760/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148318173","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}