Methods and Protocols最新文献

{"title":"Development and Characterization of a Ten-Plex Assay to Measure <i>Klebsiella pneumoniae</i> Antigen-Specific IgG in Human Sera.","authors":"Luca Rovetini, Gianina Florentina Belciug, Luisa Massai, Francesca Nonne, Renzo Alfini, Heena Ranchod, Denasha L Reddy, Mariagrazia Molfetta, Davide Oldrini, Makrina Totsika, Miren Iturriza, Ziyaad Dangor, Carlo Giannelli, Shabir A Madhi, Francesca Micoli, Martina Carducci, Omar Rossi","doi":"10.3390/mps8030052","DOIUrl":"10.3390/mps8030052","url":null,"abstract":"<p><p><i>Klebsiella pneumoniae</i> is a leading cause of nosocomial infections, neonatal sepsis, and childhood mortality worldwide. A drastic rise in antibiotic-resistant isolates poses an urgent threat to humanity, and the World Health Organization (WHO) has classified this as a critical-priority antimicrobial-resistant (AMR) pathogen. Recent advancements in developing vaccines against <i>Klebsiella pneumoniae</i> have highlighted the lack of standardized assays to evaluate immunogenicity, complicating comparison among different vaccines under development and the establishment of a serological threshold of risk reduction (SToRR). Here, we describe the development of a ten-plex multiplex assay to measure IgG against capsular polysaccharides (K2, K25, K102, K149), O antigens (O1v1, O1v2, O2v1, O2v2 and O5), and a conserved protein (MrkA). A standard curve was established by pooling human sera from naturally exposed subjects and then calibrated in terms of Relative Luminex Units/mL. The assay was fully characterized in terms of specificity, precision, linearity, and repeatability. This immunoassay demonstrates performance suitable for future clinical trials, as well as to perform sero-epidemiological studies to gain insights into naturally occurring immunity, potentially contributing to the establishment of a serological threshold of risk reduction against <i>Klebsiella pneumoniae</i>.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 3","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12101422/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144128188","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 Bioelectrical Impedance-Based Equations to Estimate Resting Metabolic Rate in Young Athletes.","authors":"Theodoros Stampoulis, Alexandra Avloniti, Dimitrios Draganidis, Dimitrios Balampanos, Polyxeni Efthimia Chalastra, Anastasia Gkachtsou, Dimitrios Pantazis, Nikolaos-Orestis Retzepis, Maria Protopapa, Athanasios Poulios, Nikolaos Zaras, Maria Michalopoulou, Ioannis G Fatouros, Athanasios Chatzinikolaou","doi":"10.3390/mps8030053","DOIUrl":"10.3390/mps8030053","url":null,"abstract":"<p><p>Resting metabolic rate (RMR) significantly impacts total daily energy expenditure, particularly on training days, and varies among trained individuals. Studies estimating RMR in this population show notable discrepancies. This study aimed to develop and validate new bioelectrical impedance analysis-based (BIA) RMR equations for young athletes, using a calibration and a validation group of 219 and 51 participants, respectively. RMR was measured via indirect calorimetry, while body composition was assessed through DXA and BIA. Correlation and agreement were evaluated by using Pearson's correlation coefficients and Bland-Altman analysis. Multiple linear regression was applied for the estimation of RMR and a one-way ANOVA was used to compare the new BIA-based equations with other specific formulas. A significant correlation was noted between the BIA and DXA measurements. The final equation, applicable to both genders, was significantly correlated with intracellular water (ICW) and trunk fat, predicting 71.1% of RMR variance. When analyzed separately, body weight and protein displayed a moderate correlation with RMR in men (r = 0.616, <i>p</i> < 0.001), while ICW was correlated with the percentage of body fat in women (r = 0.579, <i>p</i> < 0.001). In the validation group, the values obtained through the three BIA-based equations were similar to the measured RMR, but differed significantly from those obtained through the four existing equations for trained individuals. In conclusion, the developed equations based on BIA-mediated body composition analysis provide a reliable method for estimating RMR in trained populations daily.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 3","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12101231/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144128197","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 Method of Well-Spread Pachytene Chromosome Preparations for Plant Species with Large Genomes Suitable for the Immunolocalization of Meiotic Proteins.","authors":"Natalya Kudryavtseva, Aleksey Ermolaev, Ludmila Khrustaleva","doi":"10.3390/mps8030054","DOIUrl":"10.3390/mps8030054","url":null,"abstract":"<p><p>Well-spread pachytene chromosomes are critical for studying the location of meiotic proteins along individual chromosomes. However, producing good spreads in species with large genomes is challenging due to the tangling of pachytene chromosomes. Existing protocols often fail to achieve proper separation of large chromosomes in spreads. Here, we describe in detail an improved protocol that ensures the effective separation of large pachytene chromosomes and demonstrates its suitability for protein immunodetection. To develop the protocol, pollen mother cells at the middle-late pachytene stage from <i>Allium fistulosum</i>, a species with a large genome and chromosomes, were used. The protocol involved three main steps: fixing anthers in Clark's solution (ethanol-acetic acid, 3:1), digestion in an enzyme mixture, and gentle squashing in 45% acetic acid. A clear ZYP1 signal on all separated chromosomes was observed. The high quality of well-spread pachytene chromosomes obtained with the modified protocol allowed for the easy extraction of individual chromosomes for more precise detection and analysis of the proteins of interest.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 3","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12101289/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144128186","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":"Safety and Efficacy of Pressurized Intra-Thoracic Aerosol Chemotherapy in Non-Small Cell Lung Cancer Pleural Carcinomatosis: Preliminary Results of a Pilot Study.","authors":"Maria Giovanna Mastromarino, Vittorio Aprile, Gianmarco Elia, Diana Bacchin, Alessandra Lenzini, Stylianos Korasidis, Marcello Carlo Ambrogi, Silvia Martina Ferrari, Poupak Fallahi, Marco Lucchi","doi":"10.3390/mps8030051","DOIUrl":"10.3390/mps8030051","url":null,"abstract":"<p><p>Pleural carcinomatosis (PC) and malignant pleural effusion (MPE) affect up to 20% of patients with non-small cell lung cancer (NSCLC) and are usually synonymous with poor prognosis. Pressurized Intra-Thoracic Aerosol Chemotherapy (PITAC) is a novel and promising technique to control MPE in PC-NSCLC. This pilot study aimed to assess the feasibility, safety, and efficacy of PITAC in terms of palliative pleurodesis and evaluate the local antineoplastic control by analyzing patient-derived primary cell cultures. From January to December 2023, seven patients underwent PITAC with tailored doses of cisplatin and doxorubicin. There were four males and three females, with a median age of 65 (IQR:19) years. No operating room contamination by aerosolized chemotherapeutics was observed. No intraoperative complications occurred, and 30-day mortality was nil. One patient developed a postoperative prolonged air leak. The median chest tube stay was 2 (IQR:2) days, and the median hospital stay was 4 (IQR:2) days. No systemic toxicity nor hypersensitivity to chemotherapeutics were observed. All patients developed effective pleurodesis in 30 days. Cell cultures obtained from biopsy of PC-NSCLC sampled before PITAC formed confluent and monolayer sheets of attached tumor cells, while after 30 min from PITAC, cultures exhibited a significant reduction in the cancer cells' growth. Effective pleurodesis was observed three and six months after surgery in all patients. PITAC is a safe and effective technique to control MPE recurrence and might revolutionize loco-regional therapy for PC-NSCLC. Further research should assess its oncological role.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 3","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12101346/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144128202","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":"Smilebright^RO-Study Protocol for a Randomized Clinical Trial to Evaluate Oral Health Interventions in Children.","authors":"Ruxandra Sava-Rosianu, Guglielmo Campus, Vlad Tiberiu Alexa, Octavia Balean, Ruxandra Sfeatcu, Alice Murariu, Alexandrina Muntean, Daniela Esian, Constantin Daguci, Simona Olaru-Posiar, Vanessa Bolchis, Antonia Ilin, Ramona Dumitrescu, Berivan Laura Rebeca Buzatu, Mariana Postolache, Nicoleta Toderas, Roxana Oancea, Daniela Jumanca, Atena Galuscan","doi":"10.3390/mps8030049","DOIUrl":"10.3390/mps8030049","url":null,"abstract":"<p><strong>Background: </strong>Oral diseases represent a constant burden for health care and socio-economic systems as they are correlated to other non-communicable diseases. The aim of the proposed intervention is to test the effect of daily tooth brushing and oral health education on the oral health status of kindergarten children.</p><p><strong>Methods: </strong>The protocol will be conducted based on a previous epidemiological survey and conducted over 24 months; it has been developed on different levels. Dental hygienists will receive specific training to deliver oral health promotion to children and nursery educators. Training will focus on tailoring key messages to the specific age at visit; this will be outlined in the care pathway and offer practical preparation for delivering interventions and a toothpaste/toothbrush scheme. It will also, involving involve offering free daily tooth brushing to every 4-6-year-old child attending nursery. Data will be collected in four kindergartens in the capital or metropolitan areas, two kindergartens each in two large cities, and one kindergarten each in four villages from different geographic areas. Procedures used to assess the outcomes of each activity will be tailored to specific outcomes. Daily tooth-brushing activities will be monitored using qualitative research. A cost analysis including the distribution of necessary materials and correct delivery of products that shows price trends and percentage differences over the time span as well as consumer price index evaluation for the given time span will be conducted. Clinical outcomes will be evaluated using the caries incidence rate; this will be calculated for each tooth as the unit of analysis and evaluated using a multi-step approach.</p><p><strong>Discussion: </strong>Downstream oral health prevention interventions, like clinical prevention and oral health promotion, aim to enhance children's quality of life. The program's goal is to progress towards upstream interventions for a more significant impact.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 3","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-05-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12101416/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144128236","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 and Efficient DNA Extraction Protocol from Peruvian Native Cotton (<i>Gossypium barbadense</i> L.) Lambayeque, Peru.","authors":"Luis Miguel Serquén Lopez, Herry Lloclla Gonzales, Wilmer Enrique Vidaurre Garcia, Ricardo Leonidas de Jesus Velez Chicoma, Mendoza Cornejo Greta","doi":"10.3390/mps8030050","DOIUrl":"10.3390/mps8030050","url":null,"abstract":"<p><p>Efficient extraction of high-quality DNA from plants is a critical challenge in molecular research, especially in species such as <i>Gossypium barbadense</i> L., native to Peru, due to the presence of inhibitors such as polysaccharides and phenolic compounds. This study presents a modified CTAB-based protocol with silica columns that is designed to overcome these limitations without the need for liquid nitrogen or expensive reagents. Native cotton samples were collected in Lambayeque, Peru, and processed using a simplified procedure that optimizes the purity and concentration of the extracted DNA. Eight cultivars of <i>G. barbadense</i> L. with colored fibers (cream, fifo, light brown, dark brown, orange-brown, reddish, fine reddish, and white) were evaluated, yielding DNA with A260/A280 ratios between 2.14 and 2.19 and A260/A230 ratios between 1.8 and 3.14; these values are higher than those obtained with the classical CTAB method. DNA quality was validated by PCR amplification using ISSR and RAPD molecular markers, which yielded clear and well-defined banding patterns. Furthermore, the extracted DNA was suitable for advanced applications, such as Sanger sequencing, by which high-quality electropherograms were obtained. The results demonstrate that the proposed protocol is an efficient, economical, and adaptable alternative for laboratories with limited resources, allowing the extraction of high-quality DNA from <i>Gossypium barbadense</i> L. and other plant species. This simplified approach facilitates the development of genetic and biotechnological research, contributing to the knowledge and valorization of the genetic resources of Peruvian native cotton.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 3","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-05-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12101301/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144128198","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":"Empagliflozin Repurposing for Lafora Disease: A Pilot Clinical Trial and Preclinical Investigation of Novel Therapeutic Targets.","authors":"Giuseppe d'Orsi, Antonella Liantonio, Paola Imbrici, Nicola Gambacorta, Giorgia Dinoi, Cosimo Damiano Altomare, Massimo Carella","doi":"10.3390/mps8030048","DOIUrl":"10.3390/mps8030048","url":null,"abstract":"<p><strong>Background: </strong>Lafora disease (LD) is an ultra-rare and fatal neurodegenerative disorder with limited therapeutic options. Current treatments primarily address symptoms, with modest efficacy in halting disease progression, thus highlighting the urgent need for novel therapeutic approaches. Gene therapy, antisense oligonucleotides, and recombinant enzymes have recently been, and still are, under investigation. Drug repurposing may offer a promising approach to identify new, possibly effective, therapies.</p><p><strong>Methods: </strong>This study aims to investigate the conditions for repurposing empagliflozin, an SGLT2 (sodium/glucose cotransporter-2) inhibitor, as a potential treatment for LD and to establish a clinical protocol. Clinical phase: This 12-month prospective observational study will assess the safety and clinical efficacy of empagliflozin in two patients with early to intermediate LD stage. The primary endpoints will include changes in the severity of epilepsy and cognitive function, while the secondary endpoints will assess motor function, global function, and autonomy. Multiple clinical and instrumental evaluations (including MRI and PET with ¹⁸F-fluorodeoxyglucose) will be performed before and during treatment. Safety monitoring will include regular clinical assessments and reports of adverse events. Preclinical phase: In silico studies (using both molecular docking calculations and reverse ligand-based screening) and in vitro cell-based assays will allow us to investigate the effects of empagliflozin (and other gliflozins) on some key targets likely implicated in LD pathogenesis, such as GLUT1, GLUT3, glycogen synthase (hGYS), and glycogen phosphorylase (GP), as suggested in the literature and digital platforms for in silico target fishing.</p><p><strong>Results: </strong>The expected outcome of this study is twofold, i.e., (i) assessing the safety and tolerability of empagliflozin in LD patients and (ii) gathering preliminary data on its potential efficacy in improving clinical and neurologic features. Additionally, the in silico and in vitro studies may provide new insights into the mechanisms through which empagliflozin may exert its therapeutic effects in LD.</p><p><strong>Conclusion: </strong>The findings of this study are expected to provide evidence in support of the repurposing of empagliflozin for the treatment of LD.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 3","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12101192/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144128262","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":"Molecular Tools for <i>Lynx</i> spp. qPCR Identification and STR-Based Individual Identification of Eurasian Lynx (<i>Lynx lynx</i>) in Forensic Casework.","authors":"Karolina Mahlerová, Johana Alaverdyan, Lenka Vaňková, Daniel Vaněk","doi":"10.3390/mps8030047","DOIUrl":"10.3390/mps8030047","url":null,"abstract":"<p><p>The Eurasian lynx (<i>Lynx lynx</i>) is listed in CITES Appendix II and is protected under the Bern Convention and the EU Habitats Directive, yet it remains a frequent target of wildlife crime, highlighting the urgent need for reliable identification methods. This study focuses on determination and DNA quantification of the <i>Lynx</i> spp. using quantitative real-time PCR (qPCR). The <i>Llynx Qplex</i> quantification multiplex system effectively distinguishes <i>Lynx</i> spp. from other Feliformia species by targeting mitochondrial and nuclear markers. Additionally, we present the results of the developmental validation of the <i>Llyn STRplex</i> system for individual identification and databasing using six STR loci. This study followed ISFG recommendations for non-human DNA testing and developmental validation guidelines. Both systems demonstrate high sensitivity (5 pg genomic DNA for <i>Llynx Qplex</i> and 30 pg of mtDNA for <i>Llyn STRplex</i>) and high specificity to <i>Lynx</i> spp., confirmed by testing against 16 related Feliformia species. Robustness was evaluated, showing sensitivity to temperature variation, and both repeatability and reproducibility were successfully tested across replicates and conditions. Given that forensic casework often involves degraded and limited biological material, molecular tools must be both sensitive and specific to ensure accurate results. Developing precise and efficient tools is essential for supporting investigations of wildlife crime involving the Eurasian lynx, as well as efforts aimed at conserving the species.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 3","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12101276/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144128282","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":"External Validation of the NECPAL CCOMS-ICO Prognostic Tool for Early Palliative Care and Mortality Prediction in Patients with Advanced Chronic Conditions: A Prospective Observational Study Protocol.","authors":"Ana Bustamante-Fermosel, Elena Díaz-Sánchez, Natalia Pavón-Muñoz, Laetitia Hennekinne, Fuensanta Gil-Gil, Helena Notario-Leo, Alicia Sánchez-Pizarro, Marta Bustamante-Vega, Juan Torres-Macho, Anabel Franco-Moreno, On Behalf Of The Compass Research Group","doi":"10.3390/mps8030045","DOIUrl":"10.3390/mps8030045","url":null,"abstract":"<p><p>Early initiation of palliative care in patients with advanced chronic conditions significantly improves their quality of care; however, variability in disease trajectories complicates such interventions' timing. The NECPAL CCOMS-ICO prognostic tool was developed as a straightforward instrument to help healthcare providers in all clinical settings promptly identify patients with advanced chronic conditions who require palliative care, thereby enhancing service planning and delivery. Its latest version, 4.0, 2021, for the first time, incorporates a patient survival estimation. Nevertheless, validation is necessary. This study aims to validate the NECPAL version 4.0 tool in an independent cohort. It is an observational, prospective study involving outpatients and hospitalized non-randomized patients at Hospital Universitario Infanta Leonor-Virgen de la Torre in Madrid, Spain, all of whom have at least one advanced chronic condition. The study is scheduled to last 6 years, including a recruitment period of 30 months starting 1 February 2024, followed by a 12-month follow-up period for each patient. This is the first prospective study designed to validate the NECPAL version 4.0 instrument. Implementing this tool would allow the identification of patients with advanced chronic conditions and unmet palliative care needs and determine the more appropriate care pathway at the proper moment.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 3","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12101327/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144128273","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":"Multiplex Immunoassay for Biomarker Profiling of Whole Blood Cell Lysates and Supernatants and Pathogen Response in Neat Whole Blood Cultures.","authors":"Irina Balan, Alejandro G Lopez, A Leslie Morrow","doi":"10.3390/mps8030046","DOIUrl":"10.3390/mps8030046","url":null,"abstract":"<p><p>Replicating in vivo conditions is essential for understanding immune responses and measuring immune biomarkers in blood. Sampling immune biomarkers in plasma or serum often fails to detect disease-relevant signals, possibly because these markers are sequestered in immune cells or extracellular vesicles. Furthermore, traditional whole blood cultures using external media may not accurately mimic the physiological environment of blood cells. To address these limitations, we developed a strategy using whole blood cell lysates and supernatants to optimize biomarker detection. Additionally, we employed neat whole blood culture methods, preserving the natural cellular and biochemical environment to assess sensitivity to immune modulators, such as lipopolysaccharide (LPS). This cost-effective approach minimizes variability and contamination risks. By utilizing Luminex multiplex immunoassays, we profiled immune biomarkers with higher sensitivity and efficiency than traditional ELISAs. Blood samples from individuals with high alcohol consumption validated our method by assessing biomarker levels before and after LPS stimulation, providing insights into intracellular responses and inflammatory pathways. This method enhances our understanding of inflammatory processes in blood cells, demonstrating the advantages of cell lysates, supernatants, and advanced multiplex assays in immunological research.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 3","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12101426/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144128194","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}