Current protocols最新文献

Multiplex immunofluorescence microscopy facilitates the quantification and spatial analysis of cellular features within tissue sections, allowing greater understanding of disease progression or the effects of drug treatment or other exposures on cellular organization and tissue structure. Recent advances in image analysis and computational methods such as machine-learning-based cell segmentation and automated cell phenotyping have enhanced the depth of information gained from these historically qualitative images. However, many spatial analysis pipelines are technically challenging or require proprietary software or hardware, limiting accessibility and reproducibility. The free and open-source software QuPath provides a novel resource for quantifying and spatially profiling multiplex images. Here, we describe a detailed primary protocol for the semi-automated spatial analysis of 2D multiplex immunofluorescent images using QuPath, which uses object and pixel classification, cell distance, and cluster measurements for spatial profiling of tissue samples. This pipeline also includes the use of training images and basic scripting for batch processing to ensure that analysis is objective and standardized within and between projects. We also provide an alternate protocol that details a pipeline modification for whole-section images, and an extended protocol that describes the use of a free, browser-based tool to complete unsupervised, rapid processing and consolidation of the spatial data provided by QuPath, with automated reporting of cell spatial plots, cell-to-object measurements, and cell clustering data. These protocols provide an accessible, standardized, and scalable method for the spatial analysis of multiplex immunofluorescence microscopy images, facilitating reproducible quantification of cellular organization and tissue structure and thereby strengthening the integration of spatial data into translational research, biomarker discovery, and mechanistic studies. © 2026 Wiley Periodicals LLC. Basic Protocol 1: QuPath image processing Alternate Protocol: Whole-section image processing Basic Protocol 2: Use of the QuPath Spatial Analysis and Visualisation Tool.

Cranial bone regeneration requires coordinated interactions among host cells, extracellular matrices, and the local biomechanical environment to restore both mineralized tissue and structural protection of the brain. Mineralized collagen-glycosaminoglycan (MC-GAG) scaffolds recapitulate key features of native bone matrix and have demonstrated osteogenic potential in preclinical calvarial defect models; yet, robust, quantitative methods are needed to evaluate defect bridging, mineral density, and microarchitectural maturation in vivo. In vivo microcomputed tomography (microCT) enables nondestructive, three-dimensional (3D) assessment of craniofacial healing with spatial detail sufficient to characterize mineral distribution and structural connectivity across large defects. Here, we describe an integrated workflow that combines scaffold implantation in a rabbit critical-sized calvarial defect model with standardized in vivo microCT imaging and 3D computational rendering. The framework includes scaffold fabrication and preparation, surgical creation of a critical-sized calvarial defect with scaffold placement, and in vivo microCT imaging at a single endpoint (6 months post-surgery). Image datasets are visualized and analyzed using reproducible segmentation software (i.e., ORS Dragonfly) to examine defect repair and extract quantitative intensity- and volume-based metrics, including mineralized tissue volume, mineral density, and microarchitectural parameters. This standardized approach supports objective, image-based comparison of scaffold designs and treatment strategies, facilitating rigorous evaluation of cranial bone regeneration and accelerating the development of bone-regenerative biomaterials. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Fabrication of mineralized collagen-based scaffolds Basic Protocol 2: Surgical creation of rabbit calvarial defects with scaffold implantation and in vivo microCT scanning Basic Protocol 3: In vivo microCT data visualization, rendering, and analysis of MC-GAG scaffolds-implanted rabbit calvarial bone defect.

The original Morris water maze has been coined the "gold standard" task for examining spatial navigation in animals. The general procedure of the maze involves a circular pool filled approximately halfway with water. An animal is then tasked with locating and recalling the position of a hidden "platform," which is submerged below the water surface in a fixed location. The platform has minimal visual presence in the pool, meaning the location of the platform must be found, learned, and recalled from memory. Recently, the task has been translated using virtual reality for use with humans (virtual Morris water task) to investigate similar cognitive mechanisms examined using the animal version of the task. However, there are multiple variations of the virtual Morris water task scattered across the human literature. These versions vary in both environmental design (e.g., different shaped arenas or platform sizes) and testing procedures (e.g., 1-min trial times or no intertrial intervals), which influence a person's ability to perform the task. While the virtual version of this task possesses the same potential to become the "gold standard" for examining spatial cognition in humans, comparing and replicating results across research labs has been incredibly difficult due the lack of standardized procedures and protocols. In this paper, we present protocols to help with the standardization of this task. We recommend practices and procedures for researching specific cognitive processes in humans, as well as reporting guidelines, recommended analyses, and expected results. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Reactive oxygen species (ROS) are highly reactive oxygen-based molecules comprising hydrogen peroxide, hydroxyl radicals, superoxide anion, and singlet oxygen. These species are produced intracellularly and play an important role in cellular signaling and metabolism. Their high reactivity damages intracellular macromolecules such as lipids and DNA. In cancer biology, ROS display a dual role: they promote cancer cell proliferation at low to moderate levels, whereas excessive accumulation overwhelms antioxidant defenses, causing oxidative stress and apoptosis. This has resulted in therapeutic strategies that selectively increase ROS in cancer cells to induce apoptosis. Vitamin D has demonstrated anti-cancer properties, with one proposed mechanism involving ROS-mediated apoptosis. This article outlines a workflow to investigate ROS-induced cellular damage by vitamin D₃ in HeLa cervical cancer cells. The study begins with quantification of ROS levels and assessment of mitochondrial membrane potential in HeLa cultures. Transmission electron microscopy is used to examine mitochondrial ultrastructure. Lipid peroxidation quantifies downstream ROS-mediated membrane and cellular injury. Antioxidant enzyme activities, including superoxide dismutase and catalase, measure cellular anti-oxidative defence capacity. Lastly, the role of ROS inhibition of AKT signaling, leading to reduced cell survival and apoptosis, is quantified by immunoblotting. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of HeLa cell cultures and treatment with vitamin D₃ Basic Protocol 2: Quantification of ROS-positive cells using the Muse Oxidative Stress assay Basic Protocol 3: Measurement of mitochondrial membrane potential using the Muse MitoPotential assay Basic Protocol 4: Ultrastructural evaluation of mitochondrial damage by transmission electron microscopy Basic Protocol 5: Evaluation of lipid damage using a human 8-iso prostaglandin F2α ELISA Basic Protocol 6: Evaluation of total superoxide dismutase activity using an activity assay kit Basic Protocol 7: Evaluation of catalase activity using an activity assay kit Basic Protocol 8: Immunoblot analysis of AKT to assess PI3K/AKT signaling.

Yeast complementation assays provide a robust in vivo platform for characterizing the permeability and pH gating of transmembrane channels. This article details a liquid culture approach to quantify urea and ammonia transport using Saccharomyces cerevisiae deletion strains. Functional complementation, evidenced by cell growth in selective medium with urea or ammonia as the sole nitrogen source, directly reports on channel activity, generating solute-specific permeability and pH-dependency profiles. We present step-by-step procedures using the bacterial urea channel HpUreI of Helicobacter pylori, including two variants (A57C and L134C) for urea permeability and HpUreI, HpUreI E177Q, and human hAQP8 for ammonia transport. By monitoring growth across a pH range, this method enables semi-quantitative comparison of channel function. The assay is cost effective, scalable to high-throughput formats, and adaptable for studying diverse solutes, protein homologs, or mutants. It also serves as an efficient pre-screening tool for affinity tag placement before in vitro characterization. Unlike in vitro reconstitution, this approach preserves native protein-lipid interactions and avoids purification artifacts, allowing direct comparison to wild-type proteins. Though less quantitatively precise than in vitro methods, it offers higher throughput and solute flexibility compared to oocyte expression systems. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Quantifying urea permeability and pH gating using a yeast complementation growth assay Alternate Protocol: Adapting the yeast complementation assay to assess ammonia permeability and pH dependency.

MicrobiomeNet (https://microbiomenet.com) is a web-based platform developed to provide functional insights into microbiome signatures using genome-scale metabolic models (GEMs). It currently hosts 12,400 GEMs and around 6 million microbial signatures. Users can start by searching microbes, metabolites, genes, or enzymes, and perform common tasks such as to characterize the metabolic capacity for a given microbe, to explore known microbial associations, as well as to understand potential metabolic interactions. This book chapter provides practical, step-by-step instructions for navigating MicrobiomeNet to obtain functional insights into individual microbes or microbial association networks. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Characterizing the Metabolic Profile of a Microbe of Interest Basic Protocol 2: Elucidating Metabolic Interactions from Microbial Associations Basic Protocol 3: Analyzing Carbohydrate-Utilization Pathways to Explain Co-Responsive Taxa Basic Protocol 4: Identifying Novel Deoxycholic Acid-Producing Gut Microbes Basic Protocol 5: Assessing the Faecalibacterium prausnitzii-Coprococcus Relationship.

Current Protocols is issuing a correction for the following protocol article.

Wang, X., Gu, Y., Luo, H., Nowak, C., Chen, R., Hui, Y., Liu, S., & Gersweller, L. (2026). Rapid, scalable, and cost-effective manufacturing of uniform non-enveloped, tag-free virus-like particles. Current Protocols, 6, e70309. doi: 10.1002/cpz1.70309

In the above-referenced article:

The corresponding author's surname has been corrected from “Gersweller” to “Gerstweiler”.

The current version online now includes this correction and may be considered the authoritative version of record.

Acoustic imaging cytometry integrates high-throughput flow cytometry with high-resolution brightfield imaging, enabling simultaneous quantitative and morphological analysis of cells. This technology employs acoustic focusing to align cells precisely within a fluidic channel, ensuring optimal presentation to an optical system that captures high-quality images at rates up to 6000 events per second. The uncolored, high-contrast images reveal detailed cellular morphology, such as size, shape, and internal structures, complementing traditional flow cytometry data like cell counts and fluorescence intensity. This dual-modality approach is transformative for clinical research, offering a comprehensive view of heterogeneous cell populations in fields such as oncology, hematology, and immunology. © 2026 Wiley Periodicals LLC.

Basic Protocol 1: Diluting whole blood for flow cytometry of red blood cells

Basic Protocol 2: Lyse-no-wash whole blood staining

Basic Protocol 3: Stain-fix-lysis for leukocyte immunophenotyping and cell cycle analysis

Basic Protocol 4: Setting up the Attune CytPix imaging cytometer

Basic Protocol 5: Manual focusing on the Attune CytPix imaging cytometer

Basic Protocol 6: Red blood cell morphological assessment

Basic Protocol 7: Analysis of cell-to-cell interactions: Cytotoxic activity and rosetting

Basic Protocol 8: Classical morphology and biomarker analysis in myelodysplastic syndromes, plasma cell disorders, and acute myeloid leukemia

Basic Protocol 9: Cell debris discrimination in cerebrospinal fluid analysis

Basic Protocol 10: Real-time single-cell analysis: Discrimination by morphological and pulse parameters

Basic Protocol 11: DNA content and hierarchical clonal heterogeneity analysis in acute myeloid leukemia

Plant-based multiomics approaches provide powerful tools for elucidating metabolic regulation, biochemical diversity, and functional responses to genetic and environmental variation. However, plant matrices pose unique analytical challenges due to their chemical complexity, high levels of secondary metabolites, and strong matrix effects that can compromise reproducibility if workflows are not carefully standardized. This article presents a comprehensive and integrated set of protocols for untargeted plant metabolomics, lipidomics, and proteomics, coupled with robust data processing, statistical analysis, and multiomics integration strategies.

The protocols describe harmonized workflows for sample collection, preparation, and analysis using gas chromatography-mass spectrometry (GC-MS)-based metabolomics, liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-qTOF-MS)-based metabolomics, liquid chromatography-mass spectrometry (LC-MS)-based lipidomics, and microflow LC-MS/MS-based proteomics. Emphasis is placed on critical parameters specific to plant matrices, including complete solvent removal prior to GC-MS derivatization, optimized MS/MS acquisition strategies for high-confidence annotation, and quality control-driven experimental design. Detailed guidance is provided for instrument maintenance, QC strategies, and prevention of analytical artifacts.

In addition, the article outlines best practices for data preprocessing, metabolite and lipid annotation, statistical analysis, pathway mapping, and integration of metabolomics with proteomics data to support biologically meaningful interpretation. Collectively, these protocols enable reproducible, high-quality plant multiomics studies and are suitable for both method development and large-scale comparative analyses across plant species, tissues, and experimental conditions. © 2026 Wiley Periodicals LLC.

Basic Protocol 1: Plant material collection, handling, and extraction processing

Support Protocol 1: Soxhlet extraction

Alternate Protocol 1: Dichloromethane (DME)-based sample preparation for lipidomics

Alternate Protocol 2: Methyl-tert-butyl ether (MTBE)-based sample preparation for lipidomics

Basic Protocol 2: GC-MS-based metabolomics analysis

Basic Protocol 3: LC-qTOF-MS-based metabolomics analysis

Basic Protocol 4: LC-MS-based lipidomics analysis

Basic Protocol 5: Microflow LC-MS/MS-based proteomics analysis

Basic Protocol 6: Multiomics data integration and statistical analysis

The cover image is based on the article Acoustic Imaging Cytometry for High-Throughput Cell Analysis by Jordi Petriz et al., https://doi.org/10.1002/cpz1.70323.