Current protocols最新文献

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

High-field magnetic resonance imaging (MRI) scanners have been the primary choice for high-quality soft tissue imaging, but use remains limited by high costs, complex siting requirements, and infrastructure demands. In contrast, preclinical MRI scanners with a low-field permanent magnet are increasingly being adopted in research due to their lower cost, portability, and ease of installation. However, their reduced intrinsic signal-to-noise ratio (SNR) and limited pulse sequence availability constrain their use for high-quality ex vivo soft tissue characterization. To overcome these limitations, we established an optimized imaging protocol using a compact 1-Tesla permanent-magnet preclinical MRI system for high-quality ex vivo soft tissue imaging. A 25-mm (length) × 23-mm (diameter) coil was employed to image both human and animal soft tissue samples. Protocol optimization focused on four key parameters, namely the number of excitations (NEX), repetition time (TR), echo time (TE), and slice thickness, using a 3D gradient-echo (3D-GRE) acquisition sequence. Increasing the NEX enhances the SNR through signal averaging, extending the TR further improves the SNR, reducing the TE provides improved tissue contrast, and thinner slices improve image resolution. A typical 3D-GRE MRI scan using the imaging protocol takes 13 hr. The approach also incorporates standardized ex vivo sample preparation, including staining with gadolinium diethylenetriaminepentaacetic acid to enhance tissue contrast, and background proton signal suppression and sample immobilization with Fluorinert or agarose. Collectively, these optimizations substantially improve the performance of low-field permanent-magnet MRI scanners for high-quality soft tissue imaging. This set of protocols provides a practical framework for researchers to expand the capabilities of low-field MRI systems, enabling more widespread access to advanced tissue characterization methods that support disease research and therapeutic development. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Tissue sample preparation with Gd-DTPA and Fluorinert

Alternate Protocol: Tissue sample preparation with Gd-DTPA and agarose

Basic Protocol 2: High-quality sample imaging with 1-Tesla compact MRI

The scientific process is inherently iterative; we never stop at a single observation but rather conduct our research again in the same or in a different context to see if we will obtain the same results. Failure to do so makes us doubt the validity of the findings and methods of our research and the study we aimed to repeat. While this is an oversimplification of the issue, such unsuccessful attempts at replicating prior research have led to what has been globally recognized as a “Reproducibility Crisis” in research. Its many causes are deeply intertwined, and its implications are complex; yet the research community has mobilized to fight it through both formal and informal initiatives. Here we provide some tools and tips that will help make the methods, data, and code more reproducible for researchers at any stage. We hope that the uptake of these good research practices will eventually lead to positive changes within the research community. © 2025 Wiley Periodicals LLC.

Immunoheterogeneity is a potential hindrance to the maximum applicability of mesenchymal stromal cells (MSCs) in biotherapeutics and regenerative medicine. The primary causes of this heterogeneity are donor variation, diversity of tissue sources, and the culture conditions under which MSCs clonally propagate. Immunoheterogeneity could manifest as functional heterogeneity within these stem cells, affecting their ability to differentiate into adult tissue lineages. The impacted healthy human third molar tooth is a rich source of MSCs, which are isolatable both from the dental pulp and the dental follicle. Dental pulp stem cells (DPSCs), though widely characterized for their multipotential lineage differentiation, demonstrate considerable immunoheterogeneity, which demands the enrichment of these stem cells post-isolation. The aim of this study is to enrich and understand the functional differences in sub-populations of MSCs towards a specific lineage. In this protocol, we have highlighted detailed, stepwise methods for sorting and culturing post-sort and optimized methods for quantifying the differentiation of sorted DPSCs towards the osteogenic lineage. Our workflow is designed with additional considerations for cell sorter availability, maintenance of cell viability during long-distance transport to a state-of-the-art facility, and optimization of post-sorting cell culture and downstream experiments. This protocol can be universally followed to understand functional differences within MSCs isolated from different tissue sources and their implications for multi-lineage differentiation. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Assessing multiparametric expression of MSC surface markers and single cell sorting for enrichment of stem cells

Support Protocol 1: Antibody panel design

Support Protocol 2: MSC culture, preparation of cell suspension, and sample staining

Support Protocol 3: Setting up controls

Support Protocol 4: Analysis and data interpretation

Basic Protocol 2: Quantitative and qualitative assessment of osteogenic differentiation of post-sorted DPSCs

Support Protocol 5: Differentiation of MSCS (pre-sort and post-sort considerations)

Support Protocol 6: Cytochemical staining and quantification

Support Protocol 7: Immunofluorescence staining

Support Protocol 8: RNA isolation and qPCR-based characterization.

The inferior colliculus (IC) is a central hub for auditory information processing that receives widespread convergent projections. The IC comprises three main subdivisions: the central nucleus of the IC (ICC), the dorsal cortex (DC), and the lateral cortex (LC). While the ICC receives primarily ascending auditory information, DC and LC receive major cortical and multisensory projections. The LC has repeated molecular motifs that govern its input-output relationships. However, because the LC is buried deep within a sulcus, it is difficult to image in behaving animals, making it challenging to answer questions about its functional organization. Here, we describe a protocol for coupling two-photon microscopy with a microprism to obtain cellular-resolution sagittal views of functional LC maps. We employed this novel approach to investigate neuronal responses to pure tones in relation to LC motifs. This method will not only provide new insights into the auditory system but will also permit imaging of hidden brain regions previously inaccessible by conventional means. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Craniotomy and implantation of the microprism.

Basic Protocol 2: Data acquisition from sound-responsive neurons.

Basic protocol 3: Confirming the microprism location.

Basic Protocol 4: Analyzing the time traces of neuronal responses and generating a best-frequency tuning Map.