Current protocols最新文献

In vitro modeling of human liver function is essential for assessing drug-induced toxicity, particularly in the context of metabolic dysfunction–associated steatotic liver disease (MASLD). However, standard liver organoid systems often lack xenobiotic-metabolizing enzyme activity and fail to replicate the pathological features of MASLD. Here, we present a method for generating functional, multi cell–type human liver organoids (HML) organoids from HepaRG cells, primary human macrophages, and LX-2 hepatic stellate cells. These three-dimensional (3D) organoids are cultured under defined conditions that support key hepatic functions. To mimic MASLD, we expose HML organoids to a mixture of stearic and oleic acids for 9 days, inducing steatosis and fibrogenic responses characteristic of the disease. These MASLD-HML organoids retain liver-specific functions and express key fibrosis markers. We further demonstrate the usefulness of this method to produce standardized organoids useful for the evaluation of drug-induced liver injury (DILI) through IC50 and benchmark dose calculations, particularly in the context of MASLD. Together, HML and MASLD-HML organoids provide a robust model for studying drug metabolism, toxicity, and adverse drug reactions in healthy and diseased liver states. © 2026 Wiley Periodicals LLC.

Basic Protocol 1: Culture of HepaRG cells

Basic Protocol 2: Culture of LX-2 cells

Basic Protocol 3: Culture of primary human unstimulated M1 macrophages

Basic Protocol 4: Organoid seeding procedure 1 in agarose molds

Basic Protocol 5: Organoid seeding procedure 2 in ultra-low attachment 96-well plate

Alternate Protocol: Organoid seeding procedure from cryopreserved cells

Basic Protocol 6: Preparation of the fatty acid mixture

Basic Protocol 7: MASLD induction

Site-directed mutagenesis is indispensable for protein, RNA, and plasmid engineering. It is ideal to carry out such mutagenesis at an efficiency close to 100%, but many current methods fail to reach this goal and thus require extensive screening efforts. We have recently optimized an innovative site-specific mutagenesis approach based on PCR with primer pairs possessing 3’-overhangs, thereby reaching the ideal efficiency of ∼100%. Such high efficiency and the incorporation of the “handshaking” feature of primer pairs with 3’-overhangs have led us to adapt this method for seamless cassette mutagenesis, thereby conferring highly efficient deletion (up to 5 kb), insertion (up to 0.4 kb) or replacement of DNA fragments. Conceptually, deletion and insertion are special cases of replacement mutations, where the respective sequences to be inserted and deleted are 0 bp. This is because replacement mutagenesis converts fragment A to fragment B; for deletion, fragment B is 0 bp in size, whereas for insertion, fragment A is 0 bp. Thus, this new method makes site-directed and cassette mutagenesis a highly efficient and reliable tool for protein, RNA and plasmid engineering in different types of biomedical research. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: P3a site-directed mutagenesis to introduce point mutations, deletions, insertions or replacements

Basic Protocol 2: Transformation of chemically competent DH5α cells to obtain bacterial colonies

Basic Protocol 3: Isolation of plasmids from bacterial colonies for sequence analysis to identify clones with designed mutations

Alternate Protocol: P3 site-directed mutagenesis to introduce deletions, insertions, or replacements

Dermatophytes are keratinophilic fungi that cause superficial infections of the skin, hair, and nails. The emergence of recalcitrant infections caused by Trichophyton mentagrophytes complex and T. indotineae, particularly in diabetic patients, has heightened the need for reproducible small-animal models to study disease pathogenesis and test antifungal therapies. Here, we describe a diabetic mouse model of dermatophytosis that faithfully mimics human infection. The protocol details (1) preparation of dermatophyte conidial inocula from laboratory or clinical isolates, (2) pharmacological induction of diabetes or immunosuppression in mice, (3) standardized infection of abraded dorsal skin, and (4) downstream assessments of fungal burden and pathology, including clinical evaluation and scoring, enumeration of colony-forming units, and histopathology. Critical parameters, troubleshooting guidance, and anticipated results are provided to ensure reproducibility. This system has been successfully applied to evaluate conventional and investigational antifungal drugs and can be readily adapted for emerging resistant species such as T. indotineae. By integrating diabetic predisposition with current epidemiological trends, this model provides a clinically relevant and cost-effective platform for dermatophyte research and antifungal development. © 2026 Wiley Periodicals LLC.

Basic Protocol 1: Preparation of dermatophyte inoculum

Basic Protocol 2: Establishment of immunosuppressed or diabetic mouse models of dermatophytosis

Fluorescence recovery after photobleaching (FRAP) is a powerful, versatile, and widely accessible tool to monitor molecular dynamics in living cells that can be performed using modern confocal microscopes. Although the basic principles of FRAP are simple, quantitative FRAP analysis requires careful experimental design, data collection, and analysis. In this article, we discuss the theoretical basis for confocal FRAP, followed by step-by-step protocols for FRAP data acquisition using a laser-scanning confocal microscope for (1) measuring the diffusion of a membrane protein, (2) measuring the diffusion of a soluble protein, and (3) analyzing intracellular trafficking. Finally, data analysis procedures are discussed, and an equation for determining the diffusion coefficient of a molecular species undergoing pure diffusion is presented. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: How to set up a FRAP experiment

Basic Protocol 2: Confocal FRAP measurements of the lateral diffusion of plasma membrane proteins and lipids

Alternate Protocol 1: Lateral diffusion measurements for a rapidly diffusing soluble protein

Alternate Protocol 2: FRAP analysis of intracellular trafficking kinetics

Basic Protocol 3: Working with FRAP data

Basic Protocol 4: Further analysis of FRAP data to obtain diffusion coefficients

Porous collagen hydrogels are widely used to model dynamic cell-extracellular matrix (ECM) interactions relevant to inflammation, wound healing, and cancer invasion. To improve the physiological relevance of such assays, it is essential to incorporate architectural ECM characteristics identified in vivo that may affect the mechanical and molecular mechanisms of cell migration, including ECM geometry, alignment, and dimensionality. We present detailed, step-by-step protocols for generating three collagen-hydrogel-based migration assays that integrate structural guidance cues, either as cleft-like deformable gaps or tunnel-like tracks, including track generation by multiphoton (MP) laser ablation. For this application, practical guidance on laser setup and integration in commonly used MP microscopes is provided. We include example applications to compare the migratory behavior of HT1080 fibrosarcoma cells, applied both as single-cell suspensions and as pre-formed spheroids, in these spatially controlled models. The data indicate that migration efficiency increases with the presence of guidance cues, highlighting the importance of such cues for modeling invasive cell behavior in 3D. These protocols provide a standardized yet adaptable framework for researchers studying ECM-guided cell migration and evaluating therapeutic strategies that target cell motility. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Under-collagen assay

Basic Protocol 2: 3D interface assay

Basic Protocol 3: 3D tissue track assay

Support Protocol 1: Imaging of guidance models by confocal microscopy

Support Protocol 2: Excitation beam adjustment

Support Protocol 3: Generation of stacks of long tracks by three common microscope types

Mesenchymal stromal/stem cells (MSCs) are promising candidates for cell therapy and regenerative medicine. However, murine bone marrow-derived MSCs (mBMSCs) are much more difficult to isolate and purify because of their high heterogeneity and very limited numbers within bone morrow. To this end, this protocol was developed to establish an appropriate method for isolation and expansion of mBMSCs for preclinical research. Cells were dissociated and isolated mainly from compact bones via an enzyme digestion method to dissociate sufficient cell pools for further expansion ex vivo. Adherent cells collected were spindle-shaped with colony-forming units, and expressed a series of surface markers, including Oct4, CD29, and Sca-1 (stem cell antigen-1). Additionally, mBMSCs possessed potent capacity to differentiate into osteoblasts, adipocytes, and chondrocytes under appropriate culture conditions. Therefore, this study provides a standardized and reliable method for quick expansion of murine BMSCs. © 2026 Wiley Periodicals LLC.

Basic Protocol: Enzymatic digestion method

The growing utility of xeno-nucleic acids (XNAs) lies in their ability to extend the reach of genetic chemistry beyond the limits imposed by natural polymers. XNAs, with their diverse chemical backbones, resist enzymatic degradation and yet retain the capacity for sequence-defined information, and have found broad applications in biotechnology. The approach described herein provides a systematic method for the transliteration between XNAs and DNAs. This article delineates the ligase-catalyzed oligonucleotide polymerization (LOOPER) process as applied to the transcription and reverse transcription of XNA libraries using T3 DNA ligase. Two complementary procedures are presented. Basic Protocol 1 details the assembly of XNA polymers through the ligase-mediated templated ligation of 5′-phosphorylated trinucleotide anticodons bearing XNA modifications, exemplified here by locked nucleic acids (LNAs). Basic Protocol 2 describes the reverse transcription of XNA sequences into cDNA using unmodified DNA 5′-phosphorylated trinucleotide anticodons. Together, these protocols enable a bidirectional exchange between DNA and chemically diverse XNA species, a prerequisite for the application of SELEX and other evolutionary methodologies to noncanonical backbones. This ligase-based framework dispenses with substrate biases that can often be present with polymerases, allowing high-fidelity transliteration (>95%) across a variety of modified nucleotides. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Ligase-catalyzed oligonucleotide polymerization (LOOPER) for XNA synthesis

Basic Protocol 2: Ligase-catalyzed oligonucleotide polymerization (LOOPER) for cDNA synthesis from XNA templates

The recent emergence of large language models (LLMs) such as ChatGPT into the public domain has transformed academic communication. While LLMs can enhance productivity and accessibility, their use in research comes with significant ethical and methodological challenges. Here, we provide suggestions on how to responsibly use LLMs when preparing scientific manuscripts based on currently available knowledge and guidelines. We first outline the potential benefits of LLMs across different stages of writing, from finding relevant literature to drafting and editing, and finally to final formatting and pre-submission checks. However, this use should be tempered with awareness of potential risks, such as “hallucinated” information, plagiarism, bias, and breaches of confidentiality and copyright. With this in mind, we clarify principles of authorship, transparency, data protection, and academic integrity in relation to LLM use, emphasizing that LLMs cannot be listed as authors and cannot replace human reasoning, interpretation, or accountability. Practical recommendations are therefore provided to help researchers verify LLM-generated content, maintain records of prompts, maintain originality, and follow institutional rules. The most important take-home point is that LLMs should primarily remain tools that support but not substitute the work of researchers in academia, as their implementation always requires human oversight. In the context of this paper, we highlight that the authors always remain accountable for the final interpretation of their findings and their representation in their manuscript. © 2026 Wiley Periodicals LLC.

Porphyrins and porphyrin precursors are derived from intermediates in the heme biosynthetic pathway. Accurate measurement of these biochemicals is important for the diagnosis and monitoring of porphyrias. The biochemical protocols described in Part 3 include measurements of porphyrin precursors and porphyrins in the urine, feces, plasma, erythrocytes, and liver and determination of specific enzyme activities in erythrocytes and other cells. The basic protocols detailed here are needed for cost-effective first-line testing (i.e., screening) that emphasizes sensitivity for detecting or ruling out porphyrias in patients presenting with suggestive symptoms. They are also necessary for the more extensive testing that follows when first-line testing is positive. © 2026 Wiley Periodicals LLC.

Basic Protocol 1: Measuring delta-aminolevulinic acid in urine samples

Basic Protocol 2: Measuring porphobilinogen in urine samples

Basic Protocol 3: Measuring total porphyrins in urine samples

Basic Protocol 4: Measuring total porphyrins in fecal samples

Basic Protocol 5: Measuring total porphyrins in plasma and recording fluorescence scan

Basic Protocol 6: Measuring porphobilinogen in serum samples

Basic Protocol 7: Measuring total, metal-free, and zinc protoporphyrin in erythrocytes

Basic Protocol 8: Measuring porphobilinogen deaminase enzyme activity in erythrocytes

Basic Protocol 9: Measuring uroporphyrinogen decarboxylase enzyme activity in erythrocytes

Basic Protocol 10: Measuring total porphyrins in liver tissues

Honeybees (Apis mellifera) rely on olfaction for key behaviors, such as foraging and communication, making them valuable models for studying odor-guided behavior and learning. Traditional paradigms like the proboscis extension response (PER) have been used to investigate associative olfactory learning. However, these protocols typically involve manual odor delivery and visual scoring, which can limit precision and reproducibility. This article presents an automated version of the PER assay using Arduino microcontrollers for odor delivery, Bonsai software for real-time synchronization devices, video recording, and DeepLabCut (DLC) for behavioral tracking. Most hardware components, including bee holders and structural parts, are custom-designed and 3D printed, making the setup cost-effective and easily replicable. The system enables high-resolution objective quantification of responses, such as proboscis and antennal movements. Compared to manual methods, this open-source, modular setup improves throughput, standardization, and analytical accuracy in behavioral neuroscience research. © 2026 Wiley Periodicals LLC.

Basic Protocol 1: Building the experimental setup

Basic Protocol 2: Main circuitry assembly and software configuration

Basic Protocol 3: Preparing the animals and performing the PER training

Basic Protocol 4: From DLC video analysis to presenting your data