Nature Protocols最新文献

Mass photometry (MP) has emerged as a powerful approach to study quaternary biomolecular structure, dynamics and interactions. The capabilities of the method ultimately hinge on the ability to accurately measure the tiny optical contrast generated by individual molecules at a glass-water interface, which enables mass-resolved quantification of biomolecular mixtures. Ideally, this capability is limited only by photon shot noise, but in practice depends on additional parameters and details of the assay. Here, we focus on the key factors affecting MP performance and present simple steps that can be taken to achieve optimal MP measurements in terms of mass resolution, quantitative detection limit, reproducibility and analyte concentration range without compromising the speed and simplicity of the technique. Each sample takes <10 min to analyse, with an additionial 2 h if amination of the glass surface is desired.

Filter-aided expansion proteomics (FAXP) is a spatial proteomics approach designed for high-resolution analysis of formalin-fixed, paraffin-embedded (FFPE) tissues. Here we describe the integration of hydrogel-based tissue expansion with mass spectrometry, enabling isotropic expansion and robust protein retention while preserving spatial features. The FAXP workflow consists of several sequential steps, including tissue section dewaxing, in situ protein anchoring, hydrogel embedding, homogenization, staining, isotropic expansion, microdissection and filter-aided in-gel digestion to maximize peptide recovery. The Protocol integrates laser capture microdissection, enabling the precise isolation of single cells and subcellular components for subcellular spatial proteomics analysis. The approach achieves up to a fivefold linear expansion factor of FFPE tissue, including extracellular matrix-rich samples such as colorectal cancer, with less than 6% distortion, enabling the identification of an average of 2,368 proteins from single mouse liver nucleus shape and 3,312 proteins from single mouse liver cell shape using an Astral mass spectrometer. The method is compatible with diverse tissue types, including extracellular matrix-rich specimens, and integrates seamlessly with imaging workflows, such as immunostaining, for spatially resolved proteomic analysis. FAXP enables researchers to obtain comprehensive proteomic profiles with strong reproducibility and high sensitivity. The entire workflow takes ~27 h and requires only commercially available reagents and supplies and is thus accessible for researchers with intermediate expertise in tissue processing, microscopy and proteomics. FAXP can advance spatial proteomics-based studies, in particular of cancer heterogeneity, neurodegenerative diseases and cellular microenvironments within FFPE tissues, including archival clinical samples.

This protocol details the preparation and functionalization of viscoelastic synthetic antigen-presenting cells (APCs) for T cell activation, designed to enhance immunotherapeutic efficacy. Using a high-throughput microfluidic system and post-processing, we create cell-sized sodium alginate microbeads with tunable stiffness, viscoelasticity and surface chemistry, enabling them to better mimic the physical and activation properties of natural APCs. The protocol includes fabrication of synthetic cells with defined sizes, crosslinking strategies to achieve desirable mechanical properties, surface functionalization via click chemistry for attaching activation molecules, and characterization methods for mechanical and biochemical properties. Compared with traditional matrices or rigid microbeads, this approach allows precise control over the mechanical and biochemical features of synthetic APCs, ensuring optimal T cell activation. The resulting synthetic cells support robust T cell activation and expansion, enhance the CD8/CD4 T cell ratio, promote T memory stem cell (TMSC) formation and improve chimeric antigen receptor transduction efficiency, leading to superior tumor-killing efficacy in vitro and in vivo. Additionally, these synthetic cells can be efficiently removed from T cells after activation using simple centrifugation or calcium chelation, preserving the activated T cells. The complete protocol, including fabrication, functionalization and quality assessment, requires ~1 week to complete. Users should have experience in microfluidics, biomaterial handling, bioconjugation techniques and basic cell culture. This platform can be adapted for broader applications in immune cell engineering.

One of the challenges associated with functional magnetic resonance imaging (MRI) studies is integrating and causally linking complementary functional information, often obtained using different modalities. Achieving this integration requires synchronizing the spatiotemporal multimodal datasets without mutual interference. Here we present a protocol for integrating electrochemical measurements with functional MRI, enabling the simultaneous assessment of neurochemical dynamics and brain-wide activity. This Protocol addresses challenges such as artifact interference and hardware incompatibility by providing magnetic resonance-compatible electrode designs, synchronized data acquisition settings and detailed in vitro and in vivo procedures. Using dopamine as an example, the protocol demonstrates how to measure neurochemical signals with fast-scan cyclic voltammetry (FSCV) in a flow-cell setup or in vivo in rats during MRI scanning. These procedures are adaptable to various analytes measurable by FSCV or other electrochemical techniques, such as amperometry and aptamer-based sensing. By offering step-by-step guidance, this Protocol facilitates studies of neurovascular coupling with the neurochemical basis of large-scale brain networks in health and disease and could be adapted in clinical settings. The procedure requires expertise in MRI, FSCV and stereotaxic surgeries and can be completed in 7 days.

The functional complexity and anatomical organization of the nervous system are established during regional patterning of its embryonic precursor-the neural tube. Human pluripotent stem (hPS) cell-based models have emerged as valuable complements to animal models for studying neural development. Here we present the design and implementation of a microfluidic gradient device for modeling human neural tube formation and regional patterning with hPS cells. The microfluidic device enables the formation of tubular or spherical colonies of hPS cells at prescribed locations within microfluidic channels, allowing the cell colonies to form lumenal structures while being exposed to well-controlled chemical gradients for rostral-caudal and/or dorsal-ventral patterning, resulting in the formation of a microfluidic neural tube-like structure (μNTLS) or a forebrain-like structure (μFBLS). The μNTLS recapitulates important hallmarks of early human neural development, including well-defined lumenal morphologies, spatially organized regional marker expression, emergence of secondary signaling centers and the development of neural crest cells. The dorsal-ventral patterned μFBLS further recapitulates spatially segregated dorsal and ventral regions, as well as the layered segregation of early neurons from neural progenitors, mimicking human forebrain pallium and subpallium development. Both the μNTLS and μFBLS are compatible with long-term culture, live imaging, immunofluorescence staining and single-cell sequencing, serving as robust systems for studying human neurodevelopment and disease. This protocol can be implemented by a researcher with polydimethylsiloxane soft lithography and cell culture experience and takes ~8-41 d to complete, depending on the types of neural structure to model and their developmental stages, with an option for prolonged culture to promote neuronal maturation.

CRISPR screens have revolutionized the study of diverse biological processes, particularly in cancer research. Both pooled and arrayed CRISPR screens have facilitated the identification of essential genes for cell survival and proliferation, drivers of drug resistance and synthetic lethal interactions. However, applying loss-of-function CRISPR screening to non-proliferative states remains challenging, largely because of slower editing and the poor sensitivity of identifying guide RNAs that 'drop out' in a population of non-dividing cells. Here, we present a detailed protocol to accomplish this, using an inducible Cas9 system that offers precise temporal control over Cas9 expression. This inducible system allows gene editing to occur only after the non-proliferative state is fully established. We describe the complete procedure for generating an inducible Cas9-expressing model and for measuring editing efficiency by using flow cytometry. In addition, we discuss how to optimize key parameters for performing successful CRISPR screens in various non-proliferative states. We describe a detailed workflow for performing a screen in senescent cells to identify senolytic targets. This protocol is accessible to researchers with experience in molecular biology techniques and can be completed in 8-12 weeks, from the generation of an inducible Cas9 cell line clone to the analysis of a CRISPR screen for hit identification. These techniques can be applied by researchers across different fields, including stem cell differentiation, immune cell development, aging and cancer research.

Transcranial electrical stimulation (tES) has gained substantial momentum as a research and therapeutic tool; however, it suffers from challenges related to reproducibility and quality assessment due to the absence of standardized reporting practices. Here we aim to develop a comprehensive and consensus-based checklist for conducting and reporting tES studies to enhance the quality of research and reports. In this Consensus Statement, we used a Delphi approach conducted across three rounds and involving 38 experts to identify crucial elements required to report in tES studies. This consensus-driven approach included the evaluation of the interquartile deviation (>1.00), the percentage of positive responses (above 60%) and mean importance ratings (<3), hence ensuring the creation of a robust and well-balanced checklist. These metrics were utilized to assess both the consensus reached and importance ratings for each item. Consensus was reached, leading to the retention of 66 out of the initial 70 items. These items were categorized into five groups: participants (12 items), stimulation device (9 items), electrodes (12 items), current (12 items) and procedure (25 items). We then distilled a shorter version of the checklist, which includes the 26 items deemed essential. The Report Approval for Transcranial Electrical Stimulation (RATES) checklist is relevant to those carrying out and assessing tES studies, as it provides a structured framework for researchers to consider and report. For reviewers, it can serve as a tool to assess completeness, comprehensiveness and transparency of reports. In addition, the RATES checklist aims to promote a deeper understanding of tES and facilitates comparisons between studies within the field. Overall, the RATES checklist provides a shared reference point that may improve research quality, foster harmonization in reporting and, ultimately, enhance the interpretability and reproducibility of findings in both research and clinical contexts.