Nature Protocols最新文献_第7页

A multifunctional sensor for cell traction force, matrix remodeling and biomechanical assays in self-assembled 3D tissues in vitro. 一种多功能传感器，用于体外自组装3D组织的细胞牵引力、基质重塑和生物力学分析。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-01-24 DOI: 10.1038/s41596-024-01106-8

Bashar Emon, Md Saddam Hossain Joy, William C Drennan, M Taher A Saif

{"title":"A multifunctional sensor for cell traction force, matrix remodeling and biomechanical assays in self-assembled 3D tissues in vitro.","authors":"Bashar Emon, Md Saddam Hossain Joy, William C Drennan, M Taher A Saif","doi":"10.1038/s41596-024-01106-8","DOIUrl":"https://doi.org/10.1038/s41596-024-01106-8","url":null,"abstract":"Cell-matrix interactions, mediated by cellular force and matrix remodeling, result in dynamic reciprocity that drives numerous biological processes and disease progression. Currently, there is no available method for directly quantifying cell traction force and matrix remodeling in three-dimensional matrices as a function of time. To address this long-standing need, we developed a high-resolution microfabricated device that enables longitudinal measurement of cell force, matrix stiffness and the application of mechanical stimulation (tension or compression) to cells. Here a specimen comprising of cells and matrix self-assembles and self-integrates with the sensor. With primary fibroblasts, cancer cells and neurons we have demonstrated the feasibility of the sensor by measuring single or multiple cell force with a resolution of 1 nN and changes in tissue stiffness due to matrix remodeling by the cells. The sensor can also potentially be translated into a high-throughput system for clinical assays such as patient-specific drug and phenotypic screening. We present the detailed protocol for manufacturing the sensors, preparing experimental setup, developing assays with different tissues and for imaging and analyzing the data. Apart from microfabrication of the molds in a cleanroom (one time operation), this protocol does not require any specialized skillset and can be completed within 4-5 h.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143039181","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Photothermal nanofiber-mediated photoporation for gentle and efficient intracellular delivery of macromolecules. 光热纳米纤维介导的温和高效的大分子细胞内递送的光穿孔。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-01-22 DOI: 10.1038/s41596-024-01115-7

Dongyang Miao, Yuanyuan Song, Stijn De Munter, Huining Xiao, Bart Vandekerckhove, Stefaan C De Smedt, Chaobo Huang, Kevin Braeckmans, Ranhua Xiong

{"title":"Photothermal nanofiber-mediated photoporation for gentle and efficient intracellular delivery of macromolecules.","authors":"Dongyang Miao, Yuanyuan Song, Stijn De Munter, Huining Xiao, Bart Vandekerckhove, Stefaan C De Smedt, Chaobo Huang, Kevin Braeckmans, Ranhua Xiong","doi":"10.1038/s41596-024-01115-7","DOIUrl":"https://doi.org/10.1038/s41596-024-01115-7","url":null,"abstract":"Photoporation with free photothermal nanoparticles (NPs) is a promising technology for gentle delivery of functional biomacromolecules into living cells, offering great flexibility in terms of cell types and payload molecules. However, the translational use of photoporation, such as for transfecting patient-derived cells for cell therapies, is hampered by safety and regulatory concerns as it relies on direct contact between cells and photothermal NPs. A solution is to embed the photothermal NPs in electrospun nanofibers, which form a substrate for cell culture. Here we present a protocol for photothermal electrospun nanofiber (PEN)-mediated photoporation that induces membrane permeabilization by photothermal effects and enables efficient intracellular delivery of payload molecules into various cell types. By incorporating photothermal NPs within biocompatible electrospun nanofibers, direct cellular contact with NPs is avoided, thus largely mitigating safety or regulatory issues. Importantly, PEN photoporation is gentler to cells compared with electroporation, the most commonly used physical transfection method, resulting in higher-quality genetically engineered cells with better therapeutic potential. According to this protocol, it takes 2-3 d to prepare PEN culture wells with the desired cells, 3-4 d to optimize PEN photoporation parameters for intracellular delivery of payload molecules into different cell types in vitro and 4-5 weeks to evaluate the in vivo therapeutic efficacy of PEN-photoporated T cells. The protocol also provides details on how to construct the laser-based setup for performing photoporation experiments.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143024023","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Fabrication of nonplanar tapered fibers to integrate optical and electrical signals for neural interfaces in vivo. 非平面锥形光纤在体内神经界面集成光、电信号的制备。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-01-22 DOI: 10.1038/s41596-024-01105-9

Antonio Balena, Marco Bianco, Maria Samuela Andriani, Cinzia Montinaro, Barbara Spagnolo, Marco Pisanello, Filippo Pisano, Bernardo L Sabatini, Massimo De Vittorio, Ferruccio Pisanello

{"title":"Fabrication of nonplanar tapered fibers to integrate optical and electrical signals for neural interfaces in vivo.","authors":"Antonio Balena, Marco Bianco, Maria Samuela Andriani, Cinzia Montinaro, Barbara Spagnolo, Marco Pisanello, Filippo Pisano, Bernardo L Sabatini, Massimo De Vittorio, Ferruccio Pisanello","doi":"10.1038/s41596-024-01105-9","DOIUrl":"https://doi.org/10.1038/s41596-024-01105-9","url":null,"abstract":"Implantable multifunctional probes have transformed neuroscience research, offering access to multifaceted brain activity that was previously unattainable. Typically, simultaneous access to both optical and electrical signals requires separate probes, while their integration into a single device can result in the emergence of photogenerated electrical artifacts, affecting the quality of high-frequency neural recordings. Among the nontrivial strategies aimed at the realization of an implantable multifunctional interface, the integration of optical and electrical capabilities on a single, minimally invasive, tapered optical fiber probe has been recently demonstrated using fibertrodes. Fibertrodes require the application of a set of planar microfabrication techniques to a nonplanar system with low and nonconstant curvature radius. Here we develop a process based on multiple conformal depositions, nonplanar two-photon lithography and chemical wet etching steps to obtain metallic patterns on the highly curved surface of the fiber taper. We detail the manufacturing, encapsulation and back end of the fibertrodes. The design of the probe can be adapted for different experimental requirements. Using the optical setup design, it is possible to perform angle selective light coupling with the fibertrodes and their implantation and use in vivo. The fabrication of fibertrodes is estimated to require 5-9 d. Nonetheless, due to the high scalability of a large part of the protocol, the manufacture of multiple fibertrodes simultaneously substantially reduces the required time for each probe. The procedure is suitable for users with expertise in microfabrication of electronics and neural recordings.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143024020","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

An accessible workflow for high-sensitivity proteomics using parallel accumulation-serial fragmentation (PASEF). 使用平行积累-序列片段（PASEF）的高灵敏度蛋白质组学可访问的工作流程。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-01-17 DOI: 10.1038/s41596-024-01104-w

Patricia Skowronek, Georg Wallmann, Maria Wahle, Sander Willems, Matthias Mann

{"title":"An accessible workflow for high-sensitivity proteomics using parallel accumulation-serial fragmentation (PASEF).","authors":"Patricia Skowronek, Georg Wallmann, Maria Wahle, Sander Willems, Matthias Mann","doi":"10.1038/s41596-024-01104-w","DOIUrl":"https://doi.org/10.1038/s41596-024-01104-w","url":null,"abstract":"Deep and accurate proteome analysis is crucial for understanding cellular processes and disease mechanisms; however, it is challenging to implement in routine settings. In this protocol, we combine a robust chromatographic platform with a high-performance mass spectrometric setup to enable routine yet in-depth proteome coverage for a broad community. This entails tip-based sample preparation and pre-formed gradients (Evosep One) combined with a trapped ion mobility time-of-flight mass spectrometer (timsTOF, Bruker). The timsTOF enables parallel accumulation-serial fragmentation (PASEF), in which ions are accumulated and separated by their ion mobility, maximizing ion usage and simplifying spectra. Combined with data-independent acquisition (DIA), it offers high peak sampling rates and near-complete ion coverage. Here, we explain how to balance quantitative accuracy, specificity, proteome coverage and sensitivity by choosing the best PASEF and DIA method parameters. The protocol describes how to set up the liquid chromatography-mass spectrometry system and enables PASEF method generation and evaluation for varied samples by using the py_diAID tool to optimally position isolation windows in the mass-to-charge and ion mobility space. Biological projects (e.g., triplicate proteome analysis in two conditions) can be performed in 3 d with ~3 h of hands-on time and minimal marginal cost. This results in reproducible quantification of 7,000 proteins in a human cancer cell line in quadruplicate 21-min injections and 29,000 phosphosites for phospho-enriched quadruplicates. Synchro-PASEF, a highly efficient, specific and novel scan mode, can be analyzed by Spectronaut or AlphaDIA, resulting in superior quantitative reproducibility because of its high sampling efficiency.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-01-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008630","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Generating allogeneic CAR-NKT cells for off-the-shelf cancer immunotherapy with genetically engineered HSP cells and feeder-free differentiation culture. 用基因工程HSP细胞和无饲料分化培养产生用于现成癌症免疫治疗的同种异体CAR-NKT细胞。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-01-17 DOI: 10.1038/s41596-024-01077-w

Yan-Ruide Li, Kuangyi Zhou, Derek Lee, Yichen Zhu, Tyler Halladay, Jiaji Yu, Yang Zhou, Zibai Lyu, Ying Fang, Yuning Chen, Sasha Semaan, Lili Yang

{"title":"Generating allogeneic CAR-NKT cells for off-the-shelf cancer immunotherapy with genetically engineered HSP cells and feeder-free differentiation culture.","authors":"Yan-Ruide Li, Kuangyi Zhou, Derek Lee, Yichen Zhu, Tyler Halladay, Jiaji Yu, Yang Zhou, Zibai Lyu, Ying Fang, Yuning Chen, Sasha Semaan, Lili Yang","doi":"10.1038/s41596-024-01077-w","DOIUrl":"https://doi.org/10.1038/s41596-024-01077-w","url":null,"abstract":"The clinical potential of current chimeric antigen receptor-engineered T (CAR-T) cell therapy is hampered by its autologous nature that poses considerable challenges in manufacturing, costs and patient selection. This spurs demand for off-the-shelf therapies. Here we introduce an ex vivo feeder-free culture method to differentiate gene-engineered hematopoietic stem and progenitor (HSP) cells into allogeneic invariant natural killer T (AlloNKT) cells and their CAR-armed derivatives (AlloCAR-NKT cells). We include detailed information on lentivirus generation and titration, as well as the five stages of ex vivo culture required to generate AlloCAR-NKT cells, including HSP cell engineering, HSP cell expansion, NKT cell differentiation, NKT cell deep differentiation and NKT cell expansion. In addition, we describe procedures for evaluating the pharmacology, antitumor efficacy and mechanism of action of AlloCAR-NKT cells. It takes ~2 weeks to generate and titrate lentiviruses and ~6 weeks to generate mature AlloCAR-NKT cells. Competence with human stem cell and T cell culture, gene engineering and flow cytometry is required for optimal results.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-01-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008632","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

PEPPI-MS: gel-based sample pre-fractionation for deep top-down and middle-down proteomics. PEPPI-MS：凝胶样品预分离，用于深层自上而下和中向下的蛋白质组学。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-01-16 DOI: 10.1038/s41596-024-01100-0

Ayako Takemori, Philipp T Kaulich, Andreas Tholey, Nobuaki Takemori

{"title":"PEPPI-MS: gel-based sample pre-fractionation for deep top-down and middle-down proteomics.","authors":"Ayako Takemori, Philipp T Kaulich, Andreas Tholey, Nobuaki Takemori","doi":"10.1038/s41596-024-01100-0","DOIUrl":"https://doi.org/10.1038/s41596-024-01100-0","url":null,"abstract":"Top-down analysis of intact proteins and middle-down analysis of proteins subjected to limited digestion require efficient detection of traces of proteoforms in samples, necessitating the reduction of sample complexity by thorough pre-fractionation of the proteome components in the sample. SDS-PAGE is a simple and inexpensive high-resolution protein-separation technique widely used in biochemical and molecular biology experiments. Although its effectiveness for sample preparation in bottom-up proteomics has been proven, establishing a method for highly efficient recovery of intact proteins from the gel matrix has long been a challenge for its implementation in top-down and middle-down proteomics. As a much-awaited solution to this problem, we present an experimental protocol for efficient proteoform fractionation from complex biological samples using passively eluting proteins from polyacrylamide gels as intact species for mass spectrometry (PEPPI-MS), a rapid method for extraction of intact proteins separated by SDS-PAGE. PEPPI-MS allows recovery of proteins below 100 kDa separated by SDS-PAGE in solution with a median efficiency of 68% within 10 min and, unlike conventional electroelution methods, requires no special equipment, contributing to a remarkably economical implementation. The entire protocol from electrophoresis to protein purification can be performed in <5 h. By combining the resulting PEPPI fraction with other protein-separation techniques, such as reversed-phase liquid chromatography and ion mobility techniques, multidimensional proteome separations for in-depth proteoform analysis can be easily achieved.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-01-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008633","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Fabrication of high-performance tin halide perovskite thin-film transistors via chemical solution-based composition engineering. 基于化学溶液的成分工程制备高性能卤化锡钙钛矿薄膜晶体管。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-01-15 DOI: 10.1038/s41596-024-01101-z

Huihui Zhu, Youjin Reo, Geonwoong Park, Wonryeol Yang, Ao Liu, Yong-Young Noh

{"title":"Fabrication of high-performance tin halide perovskite thin-film transistors via chemical solution-based composition engineering.","authors":"Huihui Zhu, Youjin Reo, Geonwoong Park, Wonryeol Yang, Ao Liu, Yong-Young Noh","doi":"10.1038/s41596-024-01101-z","DOIUrl":"https://doi.org/10.1038/s41596-024-01101-z","url":null,"abstract":"Metal halide perovskite semiconductors have attracted considerable attention because they enable the development of devices with exceptional optoelectronic and electronic properties via cost-effective and high-throughput chemical solution processes. However, challenges persist in the solution processing of perovskite films, including limited control over crystallization and the formation of defective deposits, leading to suboptimal device performance and reproducibility. Tin (Sn2+) halide perovskite holds promise for achieving high-performance thin-film transistors (TFTs) due to its intrinsic high hole mobility. Nevertheless, reliable production of high-quality Sn2+ perovskite films remains challenging due to the rapid crystallization compared with more extensively studied lead (Pb)-based materials. Recently, composition engineering has emerged as a mature and effective strategy for realizing the high-yield fabrication of Sn2+ halide perovskite thin films. This approach cannot only achieve improved TFT performance with high hole mobilities and current ratios1-6, but also enable reliable device operation with hysteresis-free character and long-term stability7-12. Here we provide the experimental procedure for precursor preparation, film and device fabrication and characterization. The entire process typically takes 20-24 h. This protocol requires a basic understanding of metal halide perovskites, perovskite film coating process, standard TFT fabrication and measurement techniques.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-01-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008631","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Frequency locked whispering evanescent resonator (FLOWER) for biochemical sensing applications. 用于生化传感应用的锁频低语消失谐振器（FLOWER）。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-01-09 DOI: 10.1038/s41596-024-01096-7

Sartanee Suebka, Adley Gin, Judith Su

{"title":"Frequency locked whispering evanescent resonator (FLOWER) for biochemical sensing applications.","authors":"Sartanee Suebka, Adley Gin, Judith Su","doi":"10.1038/s41596-024-01096-7","DOIUrl":"https://doi.org/10.1038/s41596-024-01096-7","url":null,"abstract":"Sensitive, rapid and label-free biochemical sensors are needed for many applications. In this protocol, we describe biochemical detection using FLOWER (frequency locked optical whispering evanescent resonator)-a technique that we have used to detect single protein molecules in aqueous solution as well as exosomes, ribosomes and low part-per-trillion concentrations of volatile organic compounds. Whispering gallery mode microtoroid resonators confine light for extended time periods (hundreds of nanoseconds). When light circulates within the resonator, a portion of the electromagnetic field extends beyond the cavity, forming an evanescent field. This field interacts with bound analytes resulting in a change in the cavity's effective refractive index, which can be tracked by monitoring shifts in the resonance wavelength. The surface of the microtoroid can be functionalized to respond specifically to an analyte or biochemical interaction of interest. The frequency-locking feature of frequency locked optical whispering evanescent resonator means that the instruments respond to perturbations in the surface by very rapidly finding the new resonant frequency. Here we describe microtoroid fabrication (4-6 h), how to couple light into these devices using tapered optical fibers (20-40 min) and procedures for coupling antibodies as well as G-protein coupled receptors to the microtoroid's surface (from 1 h to 1 d depending on the target analyte). In addition, we describe our liquid handling perfusion system as well as the use of a rotary selector valve and custom fluidic chamber to optimize sample delivery. Step-by-step details on how to perform biosensing experiments and analyze the data are described; this takes 1-2 d.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142952016","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Sialic acid aptamer and RNA in situ hybridization-mediated proximity ligation assay for spatial imaging of glycoRNAs in single cells. 唾液酸适体和RNA原位杂交介导的糖核糖核酸空间成像的近距离连接实验。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-01-08 DOI: 10.1038/s41596-024-01103-x

Weijie Guo, Yuan Ma, Quanbing Mou, Xiangli Shao, Mingkuan Lyu, Valeria Garcia, Linggen Kong, Whitney Lewis, Zhenglin Yang, Shuya Lu, Yi Lu

{"title":"Sialic acid aptamer and RNA in situ hybridization-mediated proximity ligation assay for spatial imaging of glycoRNAs in single cells.","authors":"Weijie Guo, Yuan Ma, Quanbing Mou, Xiangli Shao, Mingkuan Lyu, Valeria Garcia, Linggen Kong, Whitney Lewis, Zhenglin Yang, Shuya Lu, Yi Lu","doi":"10.1038/s41596-024-01103-x","DOIUrl":"https://doi.org/10.1038/s41596-024-01103-x","url":null,"abstract":"Glycosylated RNAs (glycoRNAs) have recently emerged as a new class of molecules of substantial interest owing to their potential roles in cellular processes and diseases. However, studying glycoRNAs is challenging owing to the lack of effective research tools including, but not limited to, imaging techniques to study the spatial distribution of glycoRNAs. Recently, we reported the development of a glycoRNA imaging technique, called sialic acid aptamer and RNA in situ hybridization-mediated proximity ligation assay (ARPLA), to visualize sialic acid-containing glycoRNAs with high sensitivity and specificity. Here we describe the experimental design principles and detailed step-by-step procedures for ARPLA-assisted glycoRNA imaging across multiple cell types. The procedure includes details for target selection, oligo design and preparation, optimized steps for RNA in situ hybridization, glycan recognition, proximity ligation, rolling circle amplification and a guideline for image acquisition and analysis. With properly designed probe sets and cells prepared, ARPLA-based glycoRNA imaging can typically be completed within 1 d by users with expertise in biochemistry and fluorescence microscopy. The ARPLA approach enables researchers to explore the spatial distribution, trafficking and functional contributions of glycoRNAs in various cellular processes.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142951995","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Lymphatic collection and cell isolation from mouse models for multiomic profiling 小鼠模型淋巴收集和细胞分离用于多组学分析。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-01-08 DOI: 10.1038/s41596-024-01081-0

Marie Sabatier, Ani Solanki, Sangeetha Thangaswamy, Pin-ji Lei, Hengbo Zhou, Meghan O’Melia, Lutz Menzel, Samir Mitri, Jessalyn M. Ubellacker

{"title":"Lymphatic collection and cell isolation from mouse models for multiomic profiling","authors":"Marie Sabatier, Ani Solanki, Sangeetha Thangaswamy, Pin-ji Lei, Hengbo Zhou, Meghan O’Melia, Lutz Menzel, Samir Mitri, Jessalyn M. Ubellacker","doi":"10.1038/s41596-024-01081-0","DOIUrl":"10.1038/s41596-024-01081-0","url":null,"abstract":"Premetastatic cancer cells often spread from the primary lesion through the lymphatic vasculature and, clinically, the presence or absence of lymph node metastases impacts treatment decisions. However, little is known about cancer progression via the lymphatic system or of the effect that the lymphatic environment has on cancer progression. This is due, in part, to the technical challenge of studying lymphatic vessels and collecting lymph fluid. Here we provide a step-by-step procedure to collect both lymph and tumor-draining lymph in mouse models of cancer metastasis. This protocol has been adapted from established methods of lymph collection and was developed specifically for the collection of lymph from tumors. The approach involves the use of mice bearing melanoma or breast cancer orthotopic tumors. After euthanasia, the cisterna chyli and the tumor are exposed and viewed using a stereo microscope. Then, a glass cannula connected to a 1 mL syringe is inserted directly into the cisterna chyli or the tumor-draining lymphatics for collection of pure lymph. These lymph samples can be used to analyze the lymph-derived cancer cells using highly sensitive multiomics approaches to investigate the impact of the lymph environment during cancer metastasis. The procedure requires 2 h per mouse to complete and is suitable for users with minimal expertise in small animal handling and use of microsurgical tools under a stereo microscope. An approach to collect lymph drained from the primary tumor in mice enables the downstream characterization of the samples using metabolomics, lipidomics and flow cytometry.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"20 4","pages":"884-901"},"PeriodicalIF":13.1,"publicationDate":"2025-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.com/articles/s41596-024-01081-0.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142952018","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0