Current protocols最新文献

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.

Chara braunii is an emerging model species for studying plant evolution and development. Various members of the Chara genus have been used for pioneering studies of electrophysiology, cytoplasmic streaming, and cell biology. However, many studies have been limited by challenges, such as overgrowth with epiphytes or non-continuous growth throughout the year. Here, we present protocols for the cultivation, germination, and in vivo staining of C. braunii NIES-1604 to overcome these limitations. We constructed the custom-made cultivation chamber equipped with the illumination unit that allows for the precise simulation of natural conditions and continuous cultivation of C. braunii NIES-1604 throughout the year. By determining the optimal stratification period for C. braunii NIES-1604 oospores, we achieved a germination rate of 26%. Further, we present fluorescence-based in vivo staining protocols for routine in vivo staining of cellular structures including the plasma membrane, cell wall, and nuclei. Altogether, our affordable method of controlled cultivation of C. braunii NIES-1604 and other procedures serve as a valuable resource for establishing Chara cultures in laboratory settings and advancing its use as a model organism. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Construction of a cultivation chamber for C. braunii NIES-1604 growth

Basic Protocol 2: Vegetative propagation of C. braunii NIES-1604

Basic Protocol 3: Germination of C. braunii NIES-1604 oospores

Basic Protocol 4: In vivo staining of C. braunii NIES-1604 with fluorescent dyes

Biomaterial-based bioinks are increasingly utilized in bioprinting to engineer three-dimensional (3D) constructs with living cells for tissue engineering and disease modeling. Among various bioinks explored, alginate-based formulations stand out due to their good biocompatibility, mild gelation conditions, tunable mechanical properties, and ease of crosslinking via divalent cations such as calcium. Despite their widespread use, standardized protocols for preparing alginate-based bioinks and characterizing bioprinted constructs have not been well documented. Our laboratory has developed and validated reproducible methods for preparing a variety of alginate-based bioinks and printing cell-laden constructs tailored for diverse applications. In this article, we present detailed step-by-step protocols covering bioink preparation and rheological characterization, extrusion-based bioprinting of cell-laden constructs, post-printing culture and co-culture techniques, printability assessment, and live/dead and immunofluorescence assays. These protocols serve as a standardized framework for the fabrication and characterization of 3D bioprinted alginate-based cell-laden constructs, thereby facilitating translational research in tissue engineering, disease modeling, and preclinical therapeutic development. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Bioink preparation

Basic Protocol 2: Bioink characterization using rheology

Basic Protocol 3: Scaffold design and bioprinting

Support Protocol: 3D-printing parameter determination

Basic Protocol 4: Printability and cell viability analyses, and immunofluorescence assay

This article mainly describes a fermentation and purification method for expressing recombinant collagen protein in Escherichia coli. The method comprises constructing engineered bacteria expressing human type III collagen and adopting a strategy of feeding in batches for high-density fermentation. The rapid proliferation of bacterial cells is promoted at 37°C, and then the culture is inoculated into a fermentation tank with different carbon sources for growth. When glycerol is used as the main carbon source, the yield of recombinant collagen protein can reach 0.25 to 0.40 g/L. The method allows exploration of the differences in recombinant collagen production with different carbon sources in order to identify the most suitable fermentation medium component. The human type III collagen produced by the method has the typical structure of collagen, with high cell adhesion and the stability of tissue structure. Therefore, it can be used as the raw material for various collagen products, especially facial fillers, dressings, freeze-dried fibers, and gels. © 2025 Wiley Periodicals LLC.

Basic Protocol: Fermentation and purification of recombinant human type III collagen expressed in Escherichia coli