Methods in molecular biology最新文献

{"title":"Application of the Comet Assay in Advanced In Vitro Models.","authors":"Elise Rundén-Pran, Naouale El Yamani, Sivakumar Murugadoss, Tanima SenGupta, Eleonora Marta Longhin, Ann-Karin Hardie Olsen, Tatiana Honza, Alexandra Misci Hudecova, Erin McFadden, Solveig Brochmann, Xiaoxiong Ma, Maria Dusinska","doi":"10.1007/978-1-0716-4976-3_16","DOIUrl":"10.1007/978-1-0716-4976-3_16","url":null,"abstract":"<p><p>The comet assay (single-cell gel electrophoresis) is a simple, cost-efficient, robust, reliable, and user-friendly method for measuring DNA damage. The in vitro comet assay can be applied in advanced in vitro mini-organ and mini-tissue models. Higher-throughput formats of the assay, such as 48/96 mini-gels on GelBond^® film, and the 12-mini-gel slide format, in combination with automated scoring, make the comet assay a valuable screening method for the genotoxic potential of chemicals. In compliance with the 3Rs to reduce, refine, and replace animal experiments, the development of new approach methods (NAMs) is an important part of the paradigm shift in toxicology toward Next Generation Risk Assessment (NGRA), based on non-animal hazard identification and characterization of chemicals. In the case of advanced cell models, the cells are often grown in three-dimensional (3D) culture, and multiple cell types representative of different organs can be co-cultivated. We demonstrate the applicability of the comet assay with commonly applied advanced models from liver, lung, breast, gut, skin, and brain. We describe both the in vitro standard alkaline version of the comet assay (ACA) which measures DNA strand breaks, and the enzyme-linked modification (ELCA) that allows detection of specific base alterations by applying lesion-specific endonucleases (e.g., formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III (Endo III) for oxidized purines and pyrimidines, respectively). We consider basic methodological issues, experimental design including treatment conditions, and the importance of including cytotoxicity testing-all of which could have an impact on and/or give biased results. Protocols are provided for both the standard 2-gel and 12-gel slide formats. Further, we address critical points that need to be taken into consideration when assessing genotoxicity. The adaptation of the comet assay to advanced models such as 3D cell cultures, co-cultures, and air-liquid interface (ALI) exposure systems marks a significant advance in genotoxicity testing. These models offer more biologically relevant contexts for measuring DNA damage and repair, leading to better risk assessment and the development of safer chemicals, such as pharmaceuticals, cosmetics, food additives, as well as influencing environmental policies.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2986 ","pages":"337-383"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145582237","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Multicolor Laser Scanning Confocal Immunofluorescence Microscopy of DNA Damage Response Biomarkers.","authors":"Julian Laubenthal, Bart Krist, Alok Dhawan, Diana Anderson, Michał R Gdula","doi":"10.1007/978-1-0716-4976-3_14","DOIUrl":"10.1007/978-1-0716-4976-3_14","url":null,"abstract":"<p><p>DNA damage through endogenous and environmental toxicants is a constant threat to both a human's ability to pass on intact genetic information to its offspring as well as in somatic cells for its own survival. To counter these threats posed by DNA damage, cells have evolved a series of highly choreographed mechanisms, collectively defined as the DNA-damage response (DDR), to sense DNA lesions, signal their presence, and mediate their repair. Thus, regular DDR signaling cascades are vital to prevent the initiation and progression of many human diseases including cancer. Consequently, quantitative assessment of DNA damage and response became an important biomarker for assessment of human health and disease risk in biomonitoring studies. However, most quantitative DNA damage biomarker techniques require dissolution of the nuclear architecture and hence loss of spatial information. Laser scanning confocal immunofluorescence microscopy (LSCIM) of three-dimensionally preserved nuclei can be, quantitative and maintain the spatial information. Here we describe the experimental protocols to quantify individual key events of the DDR cascade in three-dimensionally preserved nuclei by LSCIM with high resolution, using the simultaneous detection of Rad50 as well as phosphorylated H2AX and ATM and in somatic and germ cells as an example.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2986 ","pages":"297-311"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145582392","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Analysis of PD-L1 Transcriptional Activity by Chromatin Immunoprecipitation.","authors":"Suprataptha U Reddy, Riddhi Mehta, Ivana Vancurova","doi":"10.1007/978-1-0716-4901-5_19","DOIUrl":"10.1007/978-1-0716-4901-5_19","url":null,"abstract":"<p><p>Immune checkpoint PD-L1 was originally identified as a cell surface transmembrane protein, but recent studies have demonstrated its presence also in the nucleus and suggested its role in transcriptional regulation. Interleukin-8 (IL-8, CXCL8) is a pro-angiogenic chemokine that promotes cancer progression. We have recently shown that in ovarian cancer (OC) cells, IFNγ induces nuclear accumulation of PD-L1, which is then recruited to IL-8 promoter, resulting in increased transcription of IL-8. Since the increased expression of IL-8 induces proliferation and invasion in OC cells, understanding the role of PD-L1 in transcriptional regulation of IL-8 is important for increasing the effectiveness of PD-L1 targeting immunotherapies. In this chapter, we describe a protocol that uses chromatin immunoprecipitation (ChIP) followed by real time PCR to quantitatively measure PD-L1 recruitment to IL-8 promoter. The main points of the protocol are the ChIP analysis of PD-L1 recruitment to IL-8 promoter using antibody that specifically recognizes endogenous PD-L1, and quantitative real time PCR using primers for human IL-8 promoter spanning the transcription start site (TSS) and a region ~500 bp upstream of the TSS. Our results show that IFNγ significantly increases PD-L1 recruitment to TSS of IL-8 promoter and this recruitment is even higher to the ~500 bp upstream region containing multiple transcription factors binding sites, suggesting that PD-L1 associates with IL-8 promoter by binding to other transcription factors.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2983 ","pages":"217-229"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145889349","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Clostridioides difficile Infection in Animals.","authors":"Francisco A Uzal","doi":"10.1007/978-1-0716-5328-9_16","DOIUrl":"https://doi.org/10.1007/978-1-0716-5328-9_16","url":null,"abstract":"<p><p>Clostridioides difficile is the cause of the C. difficile infection (CDI) in humans and several other animal species. The microorganism is also found in the environment and in the gastrointestinal tract of healthy individuals of multiple animals of a variety of species. Although CDI in most domestic animals has historically been described in individuals receiving antibiotic treatment, or in the case of horse, hospitalized animals, the number of cases in which no antibiotic treatment or hospitalization association was known has been increasing over the past few years. Co-infections with other bacteria or parasites, intestinal displacements, transport, or surgical or medical treatment are also possible predisposing factors for CDI in horses. In most animals species, CDI is not age-associated. Although it has been suggested that CDI may be a zoonosis, this has not been demonstrated.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"3046 ","pages":"221-232"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147839984","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Conventional, Gnotobiotic, and Humanized Microbiota Mouse Models of C. difficile Infection.","authors":"Daniel Erickson, James Collins","doi":"10.1007/978-1-0716-5328-9_14","DOIUrl":"https://doi.org/10.1007/978-1-0716-5328-9_14","url":null,"abstract":"<p><p>Clostridioides difficile infection (CDI) remains a significant public health challenge, with murine models serving as a cornerstone for investigating host-pathogen interactions, microbiota dynamics, and therapeutic interventions. Although no single mouse model has become the standard, antibiotic-induced disruption of the gut microbiota remains the most common approach to facilitate C. difficile colonization. Here, we describe flexible and reproducible mouse models of CDI that can be adapted to diverse experimental goals, including studies of acute disease, long-term colonization, and microbiota-mediated resistance. This protocol includes detailed guidance on antibiotic pretreatment, C. difficile challenge, clinical monitoring, and microbial enumeration. We also outline procedures for microbial reconstitution in germ-free mice, enabling the study of defined or humanized microbiota. Practical considerations for minimizing environmental contamination and optimizing reproducibility are also discussed.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"3046 ","pages":"189-200"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147840037","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Dual Fluorescent Labeling for Bacterial Aggregation Assays.","authors":"Anna M Tingler, Erin C Chard, Kate Psenka, Melinda A Engevik","doi":"10.1007/978-1-0716-5328-9_10","DOIUrl":"https://doi.org/10.1007/978-1-0716-5328-9_10","url":null,"abstract":"<p><p>Clostridioides difficile is an anaerobic, spore-forming pathogen that causes severe gastrointestinal disease. During infection, C. difficile interacts with other gut microbes within the intestinal mucus layer, which potentially influences colonization and disease progression. Here, we present a fluorescence-based protocol to examine bacterial adhesion and aggregation between C. difficile and other gut bacteria, such as Fusobacterium nucleatum. In this method, C. difficile is labeled with the green fluorescent dye CFDA-SE (carboxyfluorescein diacetate succinimidyl ester) and F. nucleatum or other bacterial partners are labeled with the red fluorescent dye CellTracker™ Orange CMRA (2,6-Bis-(4-chloro-phenyl)-isonicotinic acid 4-(1-carboxy-3-methyl-butoxy)-phenyl ester.) Following labeling, bacterial cultures are co-incubated in aggregation buffer, and aggregation is assessed by fluorescence microscopy. Alternatively, the labeled bacteria can be applied to cultured epithelial cells to evaluate both aggregation and host adhesion. This approach enables direct visualization and quantification of interspecies interactions, providing valuable insights into how pathobionts exploit interbacterial adhesion to persist and thrive within polymicrobial intestinal communities.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"3046 ","pages":"131-145"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147840008","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Handling and Experimentation with Germ-Free Mice.","authors":"Camila Bernardo de Brito, Bárbara Maria de Amorim-Santos, Danielle G Souza","doi":"10.1007/978-1-0716-5019-6_1","DOIUrl":"https://doi.org/10.1007/978-1-0716-5019-6_1","url":null,"abstract":"<p><p>Immediately after birth, mammals are largely colonized by microorganisms, with the gastrointestinal tract being the most commonly colonized organ. Over the years, several studies have shown that the intestinal microbiota is important for various physiological functions of the host. Gnotobiotic animal models are frequently used to better understand how the microbiota influences health and disease scenarios. Among gnotobiotic models, germ-free (GF) animals were first used in 1895, but it was not until 60 years later that germ-free colonies were suitable for large-scale experiments. The use of GF mice is an interesting and rich tool for studying the microbiome. However, their maintenance is a complex process that needs to be done carefully. In this chapter, we describe step by step how to manage and manipulate the gut microbiota of GF mice.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2993 ","pages":"1-8"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145888339","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Useful Guide for Analysis of Biomarkers in Cancer by Fluorochrome (Luminex) Technique.","authors":"Maria Faresjö","doi":"10.1007/978-1-0716-4901-5_1","DOIUrl":"https://doi.org/10.1007/978-1-0716-4901-5_1","url":null,"abstract":"<p><p>Remarkable progress in basic, translational, and clinical cancer research has been observed during the last decade. This has opened possibilities for the development of novel diagnostics and therapeutic approaches and created opportunities for personalized medicine. Cancer biomarkers are key players in human cancer progression, both peripheral and at the site of tumor. Through reliable techniques detecting biomarkers, cancer can thus be predicted, diagnosed and progression and response to therapy can be followed. Multiplex analysis of biomarkers in small blood volumes allows for rapid quantification of large number of circulating analytes. The Luminex technique allows multiple biomarkers to be measured simultaneously in small volumes and provides a convenient and sensitive tool for the detection of large number of extracellular secreted biomarkers to be used in prediction and therapy prognosis in cancer. The technique is based on so-called microspheres (beads) that serve as a solid phase for molecular detection. These individually dyed microbeads have monoclonal antibodies directed against the biomarker of interest and allow simultaneous detection of up to hundreds of biomarkers in a dual-laser flow analyzer. Biomarkers can be detected in serum- and plasma samples as well as in cell culture supernatants from in vitro cultured and stimulated cells, e.g., peripheral blood mononuclear cells (PBMC) or cancer cell lines.The need for robust detection of biomarkers for prediction as well as outcome of cancer therapy progression is of great importance. This chapter describes the Luminex technique for detection of biomarkers associated with cancer by magnetic bead sandwich immunoassay, with focus on some important pre-analytic factors, e.g., cell separation and cryopreservation and thawing of PBMC that may affect the outcome of detection of biomarkers. The Luminex technique is thus one way to discover biomarkers to predict, prognose, and improve clinical outcome of cancer.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2983 ","pages":"3-13"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145889345","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Detection of Multi-analytes Using Luminex Multiplex Bead Immunoassay.","authors":"Md Asrarul Islam, Yamin Farabih, Sunil Kumar","doi":"10.1007/978-1-0716-4901-5_2","DOIUrl":"https://doi.org/10.1007/978-1-0716-4901-5_2","url":null,"abstract":"<p><p>The Luminex multiplex bead immunoassay enables the detection of more than 100 analytes in a sample at a time. This method utilizes magnetic beads coated with antibodies of interest to measure multi-analytes simultaneously. Here, we describe a detailed protocol for multi-analyte detection of two proteins, fatty acid binding protein 1 (FABP1) and fibroblast growth factor 19 (FGF19), both related to fat metabolism, in cell lysates of HepG2 hepatocytes.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2983 ","pages":"15-23"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145889461","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Physical Exercise as a Model for Investigation of the Inflammation Response in Mice.","authors":"Albená Nunes-Silva, Antonio Felipe Souza-Gomes, Carolina Braga de Resende, Barbara Maximino Rezende, William Antonio Gonçalves","doi":"10.1007/978-1-0716-5019-6_11","DOIUrl":"https://doi.org/10.1007/978-1-0716-5019-6_11","url":null,"abstract":"<p><p>Animal models, particularly murine protocols on physical exercise, are widely used to investigate physiological adaptations and to study, prevent, and treat chronic non-communicable diseases. Controlled treadmill running enables precise manipulation of intensity and duration, overcoming limitations of voluntary wheel running for modeling inflammation. An incremental-speed treadmill test to fatigue disrupts homeostasis and acts as an acute inflammatory stimulus, eliciting local skeletal muscle and systemic immune responses. This chapter presents a standardized physical exercise protocol in mice and subsequent tissue collection, combined with intravital microscopy of skeletal muscle microcirculation and in vitro neutrophil migration assays, to quantify leukocyte recruitment and activation as a versatile model of exercise-induced inflammation.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2993 ","pages":"145-154"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145889543","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}