Nature Protocols最新文献_第6页

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

Standardized workflow for multiplexed charge detection mass spectrometry on orbitrap analyzers. 轨道阱分析仪多路电荷检测质谱的标准化工作流程。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-01-02 DOI: 10.1038/s41596-024-01091-y

Pei Su, John P McGee, Michael A R Hollas, Ryan T Fellers, Kenneth R Durbin, Joseph B Greer, Bryan P Early, Ping F Yip, Vlad Zabrouskov, Kristina Srzentić, Michael W Senko, Philip D Compton, Neil L Kelleher, Jared O Kafader

{"title":"Standardized workflow for multiplexed charge detection mass spectrometry on orbitrap analyzers.","authors":"Pei Su, John P McGee, Michael A R Hollas, Ryan T Fellers, Kenneth R Durbin, Joseph B Greer, Bryan P Early, Ping F Yip, Vlad Zabrouskov, Kristina Srzentić, Michael W Senko, Philip D Compton, Neil L Kelleher, Jared O Kafader","doi":"10.1038/s41596-024-01091-y","DOIUrl":"10.1038/s41596-024-01091-y","url":null,"abstract":"Individual ion mass spectrometry (I2MS) is the Orbitrap-based extension of the niche mass spectrometry technique known as charge detection mass spectrometry (CDMS). While traditional CDMS analysis is performed on in-house-built instruments such as the electrostatic linear ion trap, I2MS extends CDMS analysis to Orbitrap analyzers, allowing charge detection analysis to be available to the scientific community at large. I2MS simultaneously measures the mass-to-charge ratios (m/z) and charges (z) of hundreds to thousands of individual ions within one acquisition event, creating a spectral output directly into the mass domain without the need for further spectral deconvolution. A mass distribution or 'profile' can be created for any desired sample regardless of composition or heterogeneity. To assist in reducing I2MS analysis to practice, we developed this workflow for data acquisition and subsequent data analysis, which includes (i) protein sample preparation, (ii) attenuation of ion signals to obtain individual ions, (iii) the creation of a charge-calibration curve from standard proteins with known charge states and finally (iv) producing a meaningful mass spectral output from a complex or unknown sample by using the STORIboard software. This protocol is suitable for users with prior experience in mass spectrometry and bioanalytical chemistry. First, the analysis of protein standards in native and denaturing mode is presented, setting the foundation for the analysis of complex mixtures that are intractable via traditional mass spectrometry techniques. Examples of complex mixtures included here demonstrate the relevant analysis of an intact human monoclonal antibody and its intricate glycosylation patterns.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142922170","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

A guide to building a low-cost centrifuge force microscope module for single-molecule force experiments. 用于单分子力实验的低成本离心力显微镜模块的构建指南。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2024-12-30 DOI: 10.1038/s41596-024-01102-y

Jibin Abraham Punnoose, Andrew Hayden, Chai S Kam, Ken Halvorsen

{"title":"A guide to building a low-cost centrifuge force microscope module for single-molecule force experiments.","authors":"Jibin Abraham Punnoose, Andrew Hayden, Chai S Kam, Ken Halvorsen","doi":"10.1038/s41596-024-01102-y","DOIUrl":"10.1038/s41596-024-01102-y","url":null,"abstract":"The ability to apply controlled forces to individual molecules or molecular complexes and observe their behaviors has led to many important discoveries in biology. Instruments capable of probing single-molecule forces typically cost >US$100,000, limiting the use of these techniques. The centrifuge force microscope (CFM) is a low-cost and easy-to-use instrument that enables high-throughput single-molecule studies. By combining the imaging capabilities of a microscope with the force application of a centrifuge, the CFM enables the simultaneous probing of hundreds to thousands of single-molecule interactions using tethered particles. Here we present a comprehensive set of instructions for building a CFM module that fits within a commercial benchtop centrifuge. The CFM module uses a 3D-printed housing, relies on off-the-shelf optical and electrical components, and can be built for less than US$1,000 in about 1 day. We also provide detailed instructions for setting up and running an experiment to measure force-dependent shearing of a short DNA duplex, as well as the software for CFM control and data analysis. The protocol is suitable for users with basic experience in analytical biochemistry and biophysics. The protocol enables the use of CFM-based experiments and may facilitate access to the single-molecule research field.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2024-12-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142910030","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

Author Correction: Production and use of antigen tetramers to study antigen-specific B cells. 作者更正：生产和使用抗原四聚体研究抗原特异性B细胞。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2024-12-24 DOI: 10.1038/s41596-024-01131-7

Allyssa Phelps, Diego Pazos-Castro, Francesca Urselli, Emily Grydziuszko, Olivia Mann-Delany, Allison Fang, Tina D Walker, Rangana Talpe Guruge, Jaime Tome-Amat, Araceli Diaz-Perales, Susan Waserman, Jim Boonyaratanakornkit, Manel Jordana, Justin J Taylor, Joshua F E Koenig

引用次数: 0

Preparing submicrometer crystals for electron diffraction. 制备亚微米电子衍射晶体。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2024-12-20 DOI: 10.1038/s41596-024-01098-5

Alaa Shaikhqasem, Milton T Stubbs

引用次数: 0