Current protocols最新文献

DNA replication is often challenged by endogenous and exogenous sources of DNA damage, which can stall replication forks and result in single-stranded DNA (ssDNA) gaps, double-strand breaks (DSBs), and genomic instability. Detecting DNA damage specifically in newly synthesized DNA strands is essential for understanding how eukaryotic cells respond to replication stress and continue the cell cycle progression through DNA repair or DNA damage tolerance (DDT) mechanisms. Here, we present an optimized and accessible protocol for the alkaline BrdU comet assay—a single-cell technique that combines bromodeoxyuridine (BrdU; a thymidine analog) pulse-labeling of newly synthesized DNA with the alkaline comet assay (single-cell gel electrophoresis) followed by fluorescence immunodetection. This method enables the specific detection and measurement of DNA strand breaks occurring in newly replicated DNA during and immediately after the S phase, even without cell synchronization, allowing researchers to differentiate replication-associated DNA damage from overall genomic damage. We provide detailed instructions for performing the assay using human cells (RPE-1 h-TERT TP53 KO) in vitro after exposure to DNA replication-stress-inducing agents, such as hydroxyurea (HU) and ultraviolet-C (UV-C) radiation. We also demonstrate its application in translesion-synthesis-deficient (Pol eta-deficient) human fibroblasts in vitro. Importantly, this protocol supports time-course chase experiments (e.g., 0, 1, 2, and 4 hr post-treatment) to monitor the kinetics of DNA damage in nascent DNA strands. This BrdU-based protocol offers high specificity, single-cell resolution, and cost-effectiveness, making it particularly valuable for laboratories studying replication stress, post-replication DNA repair proficiency, DDT, and/or genotoxic responses in unsynchronized human cells in vitro. This protocol also adheres to the Minimum Information for Reporting Comet Assay (MIRCA) guidelines and is aligned with the objectives of the International Comet Assay Working Group (ICAW), ensuring high reproducibility and standardization. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Alkaline BrdU comet assay to assess replication-associated DNA damage in unsynchronized human cells (RPE-1 h-TERT TP53 KO) in vitro

Alternate Protocol 1: Alkaline BrdU comet assay to monitor replication-associated DNA damage dynamics in unsynchronized human cells (RPE-1 h-TERT TP53 KO) after HU and UV-C exposure and 0- to 2- hr chase

Alternate Protocol 2: Alkaline BrdU comet assay to compare replication-associated DNA damage dynamics in translesion synthesis polymerase η-deficient and complemented (XP-V comp) unsynchronized fibroblasts after UV-C exposure and 0- to 4-hr chase

To establish a standardized technical framework for large-scale production of recombinant collagen, we have employed Pichia pastoris GS115 as a host expression system and optimized the fermentation and purification processes for a transdermal peptide–type III collagen fusion protein (transdermal peptide–hCOL3A) at the 15-L bioreactor scale. A recombinant vector encoding the fusion gene was constructed through codon optimization and transformed into the GS115 strain. The seed culture was prepared via shake-flask cultivation in BMGY medium. High-cell-density fed-batch fermentation was conducted in a 15-L fermenter with an inoculum size ranging from 3% to 10%, achieving a final recombinant protein yield of 1.9 g/L. In the downstream purification, optimization of membrane-based concentration parameters and the incorporation of strong cation-exchange chromatography using an SP column enhanced the recovery rate of the target protein to 68%. Gel-filtration analysis demonstrated that transdermal peptide–hCOL3A exists as a trimer, and a CCK-8 assay revealed that it promotes proliferation of HaCaT cells in vitro, indicating biological functionality. This work successfully establishes a robust and scalable collagen production process suitable for pilot-scale manufacturing and provides a solid technical foundation for future industrial-scale translation. © 2025 Wiley Periodicals LLC.

Cancer drug resistance, whether intrinsic or acquired, underlies most relapses and treatment failures. Reliable preclinical models are crucial to define resistance mechanisms and test counterstrategies. This overview article concisely compares three model classes: (1) clinical, patient‑derived xenografts that retain tumor heterogeneity and can mirror patient resistance; (2) induced‑resistance models produced by prolonged drug selection in vivo or in vitro that recapitulate tumor evolution under therapy; and (3) engineered isogenic cell lines that isolate specific resistance drivers. We summarize some key resistance mechanisms revealed and potential therapeutic approaches informed by these models, including rational combinations, mutation‑targeted inhibitors/degraders, efflux/epigenetic modulators, and immune-related combinations. Each model has trade‑offs, but integrating them accelerates mechanistic insight and translational drug development. This overview guides selection of preclinical models and design of strategies to overcome cancer drug resistance. © 2025 Wiley Periodicals LLC.

Rheumatoid arthritis (RA) is a chronic disease involving inflammation of the joints. While the etiology of RA remains unknown, evidence exists for a significant contribution of the major histocompatibility complex (MHC). Environmental factors are also indicated as playing an etiological role. Since it is impossible to define the mechanisms contributing to disease onset and progression in humans, mouse models have been used widely. While many animal models have been generated by immunization with self-proteins to induce arthritis, type II collagen (CII)-induced arthritis is one of the most commonly used models utilized to understand the immunopathology of RA. CII constitutes 80% to 90% of total collagen content of hyaline cartilage found in joints and is a genetically conserved sequestered protein. Immunization with heterologous CII with an adjuvant in mice leads to cellular and humoral responses to heterologous and autoreactive CII-specific responses and collagen-induced arthritis (CIA). Mice immunized with CII develop inflammatory arthritis that shares many similarities in clinical, serological, and radiological features with RA in humans. However, selecting an antigen for inducing arthritis and the mouse strain are important, as not all strains are susceptible to CIA. A critical difference between RA and CIA is that in mice that lack the expression of human MHC II, the development of CIA is linked to the H2A locus, which is the homologue of HLA-DQ, while most human studies have linked RA susceptibility with HLA-DR alleles. Mice expressing HLA-DQ and HLA-DR molecules have been used to understand the role of MHC genes in susceptibility to RA. The protocols for inducing CIA in the HLA expressing transgenic mice described in this article can be used to understand how the different HLA molecules confer susceptibility to RA. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Use of mouse strains and humanized mice for modeling rheumatoid arthritis

Basic Protocol 2: Use of humanized mice for therapeutic protocols

Tumor-treating fields (TTFields) represent an innovative approach to cancer treatment that leverages low-intensity (1-3 V/cm) alternating electric fields operating at intermediate frequencies (100-300 kHz). These electric fields are specifically designed to disrupt the mitotic process, thereby inhibiting the proliferation of malignant cells. Recent research has shown that TTFields not only induce direct cytotoxic effects but also modulate the immune system by triggering immunogenic cancer cell death, thereby enhancing immune cell infiltration into tumor sites. This dual mechanism opens up new possibilities for synergistic integration with immunotherapy, which has already revolutionized oncology through the advent of immune-checkpoint inhibitors, adoptive cell therapies, and cancer vaccines. Given the immune-modulatory properties of TTFields, there is growing interest in exploring their potential synergistic effect to enhance immunotherapeutic efficacy. Recognizing the complementary role of TTFields in cancer treatment paves the way for innovative combinatorial strategies that may further improve patient outcomes by enhancing the effectiveness of both TTFields and immunotherapy. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

The lung is a structurally and immunologically complex organ, constantly exposed to airborne microbes, allergens, and pollutants. Understanding how diverse pulmonary immune cells respond to these challenges is critical for advancing respiratory disease research and identifying appropriate therapeutic interventions. Flow cytometry remains a cornerstone of immune profiling, and advances in high-parameter spectral cytometry have significantly expanded its analytical capabilities. However, challenges, such as poor tissue dissociation, spectral overlap, and loss of spatial information, can hinder the comprehensive interpretation of the lung environment. To address these limitations, we developed an optimized spectral flow cytometry platform, utilizing 5-laser Cytek Aurora spectral cytometers, for deep immunophenotyping of murine lung tissue. Our approach integrates in vivo CD45 antibody labeling—administered intravenously and oropharyngeally—to distinguish circulating, airway, and interstitial immune populations, preserving spatial context in single-cell suspensions. We utilize complementary 25+ parameter panels targeting myeloid and lymphoid compartments, built on a shared backbone to enable consistent classification across datasets. Refined tissue processing protocols support optimal recovery of representative lung cell populations, and overnight intracellular staining enhances marker resolution. Using this platform, we reliably resolve stromal, endothelial, and epithelial cells alongside immune subsets—including macrophages, monocytes, dendritic cells, eosinophils, neutrophils, T and B cells, innate lymphoid cells (ILCs), and natural killer (NK) cells—subclassified by activation, function, and tissue residency. Validation with an influenza A virus model confirmed expected dynamic immune responses and revealed previously unrecognized populations. This spatially informed approach enables high-resolution interrogation of pulmonary immunity in health and disease. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Differential in vivo labeling for spatial profiling of pulmonary immune cells by spectral cytometry

Basic Protocol 2: Preparation of single-cell suspensions from murine lung tissue for spectral cytometric analysis of immune cell populations

Basic Protocol 3: Optimized workflow and spectral flow cytometry panels for profiling of pulmonary immune cell populations in a single-cell suspension

JBrowse 2 is an open-source genome browser that provides unique features for visualizing syntenic relationships between multiple genomes. This article describes a protocol for setting up synteny views in JBrowse 2, using an assembly-to-assembly whole-genome alignment example. We detail data preparation steps, including the generation and formatting of whole-genome alignment data into formats compatible with JBrowse 2's synteny visualization capabilities, and show the GUI-driven process for setting up interactive synteny views and generating publication-quality figures. This protocol establishes methods for using JBrowse 2 to explore conserved sequences across multiple genomes. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Using JBrowse 2 to show genomic synteny

It is crucial that experiments dealing with plant–pathogen interactions are robust and reproducible. For evaluating the disease-resistance ability of a host plant against any pathogen, controlled conditions are a prerequisite that cannot be obtained with soil-based systems. Variation in environmental conditions during challenge inoculation of a pathogen, when performed in fields or a polyhouse, may lead to spurious results. This is of particular importance when dealing with disease resistance in a fruit tree like pomegranate, as determined after years of research efforts. Due to lack of uniformity and environment variability, the possibility of pathogen escapes and false positives is large under field or polyhouse conditions. To address these limitations, we have used tissue-cultured plants and developed an easy, reliable, rapid, and efficient in vitro bioassay for bacterial blight on pomegranate. Owing to its performance under a closed system, this system minimizes the risk of pathogen escapes, reduces environmental variability, and provides a robust high-throughput screening platform for disease resistance and tissue sampling for downstream functional genomics studies. The protocols enable reproducible experiments to study plant response to bacterial blight infection. Stepwise protocols and detailed instructions are provided to carry out the screening experiment in pomegranate or other similar plant species. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Preparation of nodal explants and in vitro rooting

Basic Protocol 2: In vitro blight bioassay

Basic Protocol 3: Pathogen re-isolation and confirmation

This paper, the second in a two-part series, outlines a low-cost, open-source, pseudo-randomization approach for measuring cutaneous reflexes during human walking. The protocol guides users through integrating an Arduino Uno microcontroller, a VICON data acquisition system, and an electrical stimulation device. A custom code was developed in C++, and steps are provided to guide users in downloading and integrating it with an Arduino. This setup provides researchers the ability to define key experimental parameters such as the number of gait cycles to include in quantifying the average time of the subject's walking gait cycle, the number of bins to partition the human walking gait cycle, and the number of stimuli per bin—all while maintaining real-time stimulation capability. During a walking trial, the system is designed to (1) deliver stimuli pseudo-randomly using instances when the foot makes initial contact with the treadmill surface and (2) deliver an even distribution of stimuli across all bins. Real-time feedback mechanisms and data monitoring are enabled via an integrated Arduino and VICON framework, allowing researchers to conduct controlled, repeatable experiments that aim to measure cutaneous reflexes during human walking. This protocol provides a flexible and accessible solution for researchers aiming to investigate phase-dependent modulation of reflexes during normal and perturbed walking conditions. Its modularity and integration with existing motion capture systems make it a powerful tool for both basic and clinical research within the fields of neuroscience, biomechanics, and gait rehabilitation. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Integrating Arduino to VICON data acquisition and stimulator

Basic Protocol 2: Downloading and running pseudo-randomized Arduino code

Angiogenesis is crucial in tissue repair, wound healing, and embryo development. Maintaining a balance between pro-angiogenic and anti-angiogenic factors is crucial for tissue homeostasis, thereby preventing the development of pathological conditions. In addition, the tissue microenvironment constantly influences molecular signaling, potentially altering this delicate balance. The chorioallantoic membrane (CAM) assay has emerged as a dynamic, cost-effective, and ethically favorable alternative for studying angiogenesis. This work presents a detailed and accessible approach to the CAM assay, focusing on the evaluation of the angiogenic potential of applied stimuli (such as conditioned media, extracellular vesicles, or bioscaffolds) through various techniques, including hematoxylin and eosin, PicroSirius Red, Alcian Blue, and resorcin-fuchsin staining; immunofluorescence; immunohistochemistry; and scanning electron microscopy (SEM). Our protocol uses the in ovo CAM assay approach, utilizing a simple incubator setup that eliminates the need for expensive equipment. The primary objective is to provide a comprehensive methodology for researchers to efficiently analyze angiogenesis, offering insights into vessel morphology and protein expression. The experiment comprises three major steps: (1) egg opening, stimulation, and sample collection; (2) histological processing and paraffin blocking; and (3) microscopic analyses. The first and second procedures take approximately 15 days, after which each analysis can be conducted whenever the researcher judges appropriate. The expected results include several types of data concerning the proliferation rate, vessel presence and integrity, measurement of angiogenic-specific proteins, and investigation of structural proteins. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Angiogenesis quail and chicken chorioallantoic membrane assay analyses