Pub Date : 2024-01-01DOI: 10.1007/978-1-0716-3690-9_18
Zhinous Hosseini, Christopher J Groves, Penny Anders, Kristen Cave, Madelyn Krunkosky, Brandi Chappell, Sofie Pattyn, Devin Davis, Sylvia Janetzki, Elizabeth Reap
Interferon-gamma (IFNγ) ELISpot and FluoroSpot are widely used assays to detect functional cell responses in immunotherapy clinical studies. Recognized for their importance in vaccine development studies to quantitate immune responses, these assays have more recently risen to the forefront in cell and gene therapy as well as cancer immunotherapy fields where responses against cancer neoantigens are not easily detectable above assay background. Here, we test a new class of fetal bovine serum (FBS), CultraPure FBS, in ex vivo ELISpot and FluoroSpot assays and cultured FluoroSpot assays following in vitro expansion. Several CultraPure FBS lots that have been specially formulated through the process of lyophilization (lyo-FBS) were compared to liquid CultraPure FBS. We stimulated human PBMCs with antigen-specific peptide pools diluted in media supplemented with liquid CultraPure FBS or lyo-FBS and found equivalent cytokine production with negligible to no assay background with both liquid and lyo-FBS formats. Moreover, the lyo-FBS showed lot-to-lot consistency and 90-day refrigerated (4 °C) stability in both ex vivo direct and in vitro cultured assays. In addition, we present here a method using lyo-FBS for the expansion of low-frequency antigen-specific T cells, mimicking the low frequency seen with cancer neoantigens by utilizing a cultured FluoroSpot assay. Our results demonstrate the presence of Granzyme B, interferon-gamma (IFNγ), and tumor necrosis factor (TNF) production by antigen-specific polyfunctional T cells following a 9-day culture using media supplemented with lyo-FBS.
{"title":"Performance and Stability of New Class of Fetal Bovine Sera (FBS) and Its Lyophilized Form in ELISpot and FluoroSpot Assays: Applications for Monitoring the Immune Response in Vaccine, and Cell and Gene Immunotherapy in Clinical Trials.","authors":"Zhinous Hosseini, Christopher J Groves, Penny Anders, Kristen Cave, Madelyn Krunkosky, Brandi Chappell, Sofie Pattyn, Devin Davis, Sylvia Janetzki, Elizabeth Reap","doi":"10.1007/978-1-0716-3690-9_18","DOIUrl":"https://doi.org/10.1007/978-1-0716-3690-9_18","url":null,"abstract":"<p><p>Interferon-gamma (IFNγ) ELISpot and FluoroSpot are widely used assays to detect functional cell responses in immunotherapy clinical studies. Recognized for their importance in vaccine development studies to quantitate immune responses, these assays have more recently risen to the forefront in cell and gene therapy as well as cancer immunotherapy fields where responses against cancer neoantigens are not easily detectable above assay background. Here, we test a new class of fetal bovine serum (FBS), CultraPure FBS, in ex vivo ELISpot and FluoroSpot assays and cultured FluoroSpot assays following in vitro expansion. Several CultraPure FBS lots that have been specially formulated through the process of lyophilization (lyo-FBS) were compared to liquid CultraPure FBS. We stimulated human PBMCs with antigen-specific peptide pools diluted in media supplemented with liquid CultraPure FBS or lyo-FBS and found equivalent cytokine production with negligible to no assay background with both liquid and lyo-FBS formats. Moreover, the lyo-FBS showed lot-to-lot consistency and 90-day refrigerated (4 °C) stability in both ex vivo direct and in vitro cultured assays. In addition, we present here a method using lyo-FBS for the expansion of low-frequency antigen-specific T cells, mimicking the low frequency seen with cancer neoantigens by utilizing a cultured FluoroSpot assay. Our results demonstrate the presence of Granzyme B, interferon-gamma (IFNγ), and tumor necrosis factor (TNF) production by antigen-specific polyfunctional T cells following a 9-day culture using media supplemented with lyo-FBS.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2768 ","pages":"305-316"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140175436","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-01DOI: 10.1007/978-1-0716-3746-3_13
Valeria Garcia-Flores, Derek Miller, Jose Galaz, Nardhy Gomez-Lopez
The dissociation of whole tissue into single-cell suspensions is a critical step for techniques focused on profiling of individual cells. Here, we describe a protocol for the preparation of high-quality single-cell suspensions from human placental tissues: the basal plate (BP), placental villi (PV), and chorioamniotic membranes (CAM). This protocol also provides guidance for the cryopreservation and recovery of single-cell suspensions for later use. The methods described here have been demonstrated to be suitable for downstream single-cell applications, such as single-cell RNA-sequencing, that require viable, high-quality cell suspensions.
{"title":"Dissociation of Placental Tissues for Single-Cell Techniques.","authors":"Valeria Garcia-Flores, Derek Miller, Jose Galaz, Nardhy Gomez-Lopez","doi":"10.1007/978-1-0716-3746-3_13","DOIUrl":"https://doi.org/10.1007/978-1-0716-3746-3_13","url":null,"abstract":"<p><p>The dissociation of whole tissue into single-cell suspensions is a critical step for techniques focused on profiling of individual cells. Here, we describe a protocol for the preparation of high-quality single-cell suspensions from human placental tissues: the basal plate (BP), placental villi (PV), and chorioamniotic membranes (CAM). This protocol also provides guidance for the cryopreservation and recovery of single-cell suspensions for later use. The methods described here have been demonstrated to be suitable for downstream single-cell applications, such as single-cell RNA-sequencing, that require viable, high-quality cell suspensions.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2781 ","pages":"143-154"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140175464","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-01DOI: 10.1007/978-1-0716-3746-3_10
Giovana Fernanda Cosi Bento, Márcia Guimarães da Silva, Ramkumar Menon, Lauren S Richardson
Modeling human pregnancy is challenging as two subjects, the mother and fetus, must be evaluated in tandem. To understand pregnancy, parturition, and adverse pregnancy outcomes, the two feto-maternal interfaces (FMi) that form during gestation (i.e., the placenta and fetal membrane) need to be investigated to understand their biological roles, and organ dysfunction can lead to adverse outcomes. Adverse pregnancy outcomes such as preterm rupture of the membranes, spontaneous preterm birth, preeclampsia, intra-uterine growth restriction, and gestational diabetes rates are on the rise worldwide, highlighting the need for future studies and a better understanding of molecular and cellular pathways that contribute to disease onset. Current in vivo animal models nor in vitro cell culture systems can answer these questions as they do not model the function or structure of human FMis. Utilizing microfabrication and soft-lithography techniques, microfluidic organ-on-chip (OOC) devices have been adapted by many fields to model the anatomy and biological function of complex organs and organ systems within small in vitro platforms.These techniques have been adapted to recreate the fetal membrane FMi (FMi-OOC) using immortalized cells and collagen derived from patient samples. The FMi-OOC is a four-cell culture chamber, concentric circle system, that contains both fetal (amniochorion) and maternal (decidua) cellular layers and has been validated to model physiological and pathological states of pregnancy (i.e., ascending infection, systemic oxidative stress, and maternal toxicant exposure). This platform is fully compatible with various analytical methods such as microscopy and biochemical analysis. This protocol will outline this device's fabrication, cell loading, and utility to model ascending infection-related adverse pregnancy outcomes.
{"title":"Feto-Maternal Interface Organ-on-Chip: A New Technology to Study Ascending Infection.","authors":"Giovana Fernanda Cosi Bento, Márcia Guimarães da Silva, Ramkumar Menon, Lauren S Richardson","doi":"10.1007/978-1-0716-3746-3_10","DOIUrl":"https://doi.org/10.1007/978-1-0716-3746-3_10","url":null,"abstract":"<p><p>Modeling human pregnancy is challenging as two subjects, the mother and fetus, must be evaluated in tandem. To understand pregnancy, parturition, and adverse pregnancy outcomes, the two feto-maternal interfaces (FMi) that form during gestation (i.e., the placenta and fetal membrane) need to be investigated to understand their biological roles, and organ dysfunction can lead to adverse outcomes. Adverse pregnancy outcomes such as preterm rupture of the membranes, spontaneous preterm birth, preeclampsia, intra-uterine growth restriction, and gestational diabetes rates are on the rise worldwide, highlighting the need for future studies and a better understanding of molecular and cellular pathways that contribute to disease onset. Current in vivo animal models nor in vitro cell culture systems can answer these questions as they do not model the function or structure of human FMis. Utilizing microfabrication and soft-lithography techniques, microfluidic organ-on-chip (OOC) devices have been adapted by many fields to model the anatomy and biological function of complex organs and organ systems within small in vitro platforms.These techniques have been adapted to recreate the fetal membrane FMi (FMi-OOC) using immortalized cells and collagen derived from patient samples. The FMi-OOC is a four-cell culture chamber, concentric circle system, that contains both fetal (amniochorion) and maternal (decidua) cellular layers and has been validated to model physiological and pathological states of pregnancy (i.e., ascending infection, systemic oxidative stress, and maternal toxicant exposure). This platform is fully compatible with various analytical methods such as microscopy and biochemical analysis. This protocol will outline this device's fabrication, cell loading, and utility to model ascending infection-related adverse pregnancy outcomes.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2781 ","pages":"105-117"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140175466","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-01DOI: 10.1007/978-1-0716-3766-1_17
Olga Raskina, Ofir Hakim
Fluorescence in situ hybridization (FISH) technique has been widely used to detect and localize specific DNA and RNA sequences in interphase nuclei and chromosomes in animals and plants. Here, we present a protocol for localization of genomic loci in nuclei of the model plant Arabidopsis thaliana. This protocol includes several advances and adaptations to A. thaliana, including preparation of nuclei and chromosomes without the use of liquid nitrogen, and an in situ hybridization procedure that preserves chromatin structure without the use of paraformaldehyde and formamide. Simultaneous denaturation of the BAC (bacterial artificial chromosome) probe and nuclei followed by annealing at high temperature allows hybridization in less than an hour. These hybridization conditions also provide high signal to noise ratio by a small number of washes. Thus, this simplified in situ hybridization procedure is completed in one working day.
荧光原位杂交(FISH)技术已被广泛用于检测和定位动物和植物间期细胞核和染色体中的特定 DNA 和 RNA 序列。在此,我们介绍一种在模式植物拟南芥细胞核中定位基因组位点的方案。该方案包括几项进步和对拟南芥的改良,包括无需使用液氮即可制备细胞核和染色体,以及无需使用多聚甲醛和甲酰胺即可保留染色质结构的原位杂交程序。BAC(细菌人工染色体)探针和细胞核同时变性,然后在高温下退火,杂交时间不到一小时。在这些杂交条件下,只需少量的洗涤就能获得高信噪比。因此,这种简化的原位杂交程序可在一个工作日内完成。
{"title":"Rapid DNA-FISH in Arabidopsis thaliana Somatic Cells.","authors":"Olga Raskina, Ofir Hakim","doi":"10.1007/978-1-0716-3766-1_17","DOIUrl":"https://doi.org/10.1007/978-1-0716-3766-1_17","url":null,"abstract":"<p><p>Fluorescence in situ hybridization (FISH) technique has been widely used to detect and localize specific DNA and RNA sequences in interphase nuclei and chromosomes in animals and plants. Here, we present a protocol for localization of genomic loci in nuclei of the model plant Arabidopsis thaliana. This protocol includes several advances and adaptations to A. thaliana, including preparation of nuclei and chromosomes without the use of liquid nitrogen, and an in situ hybridization procedure that preserves chromatin structure without the use of paraformaldehyde and formamide. Simultaneous denaturation of the BAC (bacterial artificial chromosome) probe and nuclei followed by annealing at high temperature allows hybridization in less than an hour. These hybridization conditions also provide high signal to noise ratio by a small number of washes. Thus, this simplified in situ hybridization procedure is completed in one working day.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2784 ","pages":"259-270"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140175519","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-01DOI: 10.1007/978-1-0716-3766-1_10
Charles J Sheehan, Kate D Meyer
N6-methyladenosine (m6A) is an abundant mRNA modification which plays important roles in regulating RNA function and gene expression. Traditional methods for visualizing mRNAs within cells cannot distinguish m6A-modified and unmodified versions of the target transcript, thus limiting our understanding of how and where methylated transcripts are localized within cells. Here, we describe DART-FISH, a visualization technique which enables simultaneous detection of both m6A-modified and unmodified target transcripts. DART-FISH combines m6A-dependent C-to-U editing with mutation-selective fluorescence in situ hybridization to specifically detect methylated and unmethylated transcript copies, enabling the investigation of m6A stoichiometry and methylated mRNA localization in single cells.
N6-甲基腺苷(m6A)是一种丰富的 mRNA 修饰,在调节 RNA 功能和基因表达方面发挥着重要作用。传统的细胞内mRNA可视化方法无法区分目标转录本的m6A修饰和未修饰版本,从而限制了我们对甲基化转录本在细胞内的定位方式和位置的了解。在这里,我们介绍一种可视化技术 DART-FISH,它能同时检测 m6A 修饰和未修饰的目标转录本。DART-FISH 将依赖于 m6A 的 C 到 U 编辑与突变选择性荧光原位杂交结合起来,特异性地检测甲基化和未甲基化的转录本拷贝,从而能够研究单细胞中 m6A 的配位和甲基化 mRNA 的定位。
{"title":"Simultaneous In Situ Detection of m<sup>6</sup>A-Modified and Unmodified RNAs Using DART-FISH.","authors":"Charles J Sheehan, Kate D Meyer","doi":"10.1007/978-1-0716-3766-1_10","DOIUrl":"https://doi.org/10.1007/978-1-0716-3766-1_10","url":null,"abstract":"<p><p>N<sup>6</sup>-methyladenosine (m<sup>6</sup>A) is an abundant mRNA modification which plays important roles in regulating RNA function and gene expression. Traditional methods for visualizing mRNAs within cells cannot distinguish m<sup>6</sup>A-modified and unmodified versions of the target transcript, thus limiting our understanding of how and where methylated transcripts are localized within cells. Here, we describe DART-FISH, a visualization technique which enables simultaneous detection of both m<sup>6</sup>A-modified and unmodified target transcripts. DART-FISH combines m<sup>6</sup>A-dependent C-to-U editing with mutation-selective fluorescence in situ hybridization to specifically detect methylated and unmethylated transcript copies, enabling the investigation of m<sup>6</sup>A stoichiometry and methylated mRNA localization in single cells.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2784 ","pages":"147-161"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140175522","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-01DOI: 10.1007/978-1-0716-3629-9_4
Z Faidon Brotzakis
The electron microscopy metainference integrative structural biology method enables the combination of cryo-electron microscopy electron density maps with molecular modeling techniques such as molecular dynamics to unveil the atomistic biomolecular structural ensemble and the error in the map data in an efficient manner. Here we illustrate the electron microscopy metainference protocol and analysis used to elucidate the atomistic structural ensemble of the microtubule-associated protein tau bound to microtubules by using state-of-the-art molecular mechanic force field and the electron density map.
{"title":"Cryo-electron Microscopy and Molecular Modeling Methods to Characterize the Dynamics of Tau Bound to Microtubules.","authors":"Z Faidon Brotzakis","doi":"10.1007/978-1-0716-3629-9_4","DOIUrl":"10.1007/978-1-0716-3629-9_4","url":null,"abstract":"<p><p>The electron microscopy metainference integrative structural biology method enables the combination of cryo-electron microscopy electron density maps with molecular modeling techniques such as molecular dynamics to unveil the atomistic biomolecular structural ensemble and the error in the map data in an efficient manner. Here we illustrate the electron microscopy metainference protocol and analysis used to elucidate the atomistic structural ensemble of the microtubule-associated protein tau bound to microtubules by using state-of-the-art molecular mechanic force field and the electron density map.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2754 ","pages":"77-90"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140184777","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-01DOI: 10.1007/978-1-0716-3629-9_27
Pierre Dourlen
Drosophila is a powerful model to study human diseases thanks to its genetic tools and ease of screening. Human genes can be expressed in targeted organs and their toxicity assessed on easily scorable external phenotypes that can be used as readouts to perform genetic screens of toxicity modifiers. In this chapter, I describe how to express human Tau protein in the Drosophila eye, assess protein expression by Western blot, assess Tau toxicity by quantifying the size of the Tau-induced rough eye, and perform a genetic screen of modifiers of Tau toxicity in the Drosophila eye.
{"title":"Identification of Tau Toxicity Modifiers in the Drosophila Eye.","authors":"Pierre Dourlen","doi":"10.1007/978-1-0716-3629-9_27","DOIUrl":"10.1007/978-1-0716-3629-9_27","url":null,"abstract":"<p><p>Drosophila is a powerful model to study human diseases thanks to its genetic tools and ease of screening. Human genes can be expressed in targeted organs and their toxicity assessed on easily scorable external phenotypes that can be used as readouts to perform genetic screens of toxicity modifiers. In this chapter, I describe how to express human Tau protein in the Drosophila eye, assess protein expression by Western blot, assess Tau toxicity by quantifying the size of the Tau-induced rough eye, and perform a genetic screen of modifiers of Tau toxicity in the Drosophila eye.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2754 ","pages":"483-498"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140184784","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-01DOI: 10.1007/978-1-0716-3629-9_33
Nadine Allroggen, Helen Breuer, Sarah Bachmann, Michael Bell, Hans Zempel
The study of microtubule (MT) dynamics is essential for the understanding of cellular transport, cell polarity, axon formation, and other neurodevelopmental mechanisms. All these processes rely on the constant transition between assembly and disassembly of tubulin polymers to/from MTs, known as dynamic instability. This process is well-regulated, among others, by phosphorylation of microtubule-associated proteins (MAP), including the Tau protein. Protein kinases, in particular the microtubule affinity regulating kinase (MARK), regulate the MT-Tau interaction, inducing Tau dissociation by phosphorylation. Phosphorylated Tau dissociates from microtubules forming insoluble aggregates known as neurofibrillary tangles. These accumulations of hyperphosphorylated Tau in the neurons disrupt the physiological MT-based transport machinery within the cell and can potentially lead to the development of neurodegenerative disorders, such as Alzheimer's disease (AD) and related tauopathies. Further investigations on the MT cytoskeleton dynamics are essential as they may elucidate pathomechanisms of neurodegenerative diseases - particularly tauopathies - as well as fundamental neurodevelopmental processes.The study of the dynamic assembly and disassembly of the MT network requires live-cell imaging rather than conventional immunocytochemistry based on fixed samples. To investigate MT dynamics, we perform live-cell imaging of neurons transfected with a fluorescently tagged version of the microtubule plus-end tracking protein (+TIP) EB3. This protein associates with the growing ends of MTs and thus visualizes MT growth in real time. Our imaging analysis protocol allows the determination of quantity, orientation, and velocity of MT growth in the soma and neurites of transfected neurons, using ImageJ-based tracking software and kymographs. Furthermore, functional effects of Tau and MARK kinases on the MT cytoskeleton can be assessed by overexpression or downregulation experiments of the respective protein prior to the live imaging assay. We use two different human neuronal cell models, naive and differentiated SH-SY5Y neuroblastoma cells, and neurons derived from induced pluripotent stem cells (iPSCs), both of which have shown success as models to study Tau-related pathologies.This protocol describes an optimized method for analysis of microtubule dynamics using fluorescent tagged EB3 protein as microtubule plus end marker. In this chapter, we outline the process of neuronal transfection, live-cell imaging, and necessary time-lapse image analysis based on ImageJ in two human-derived neuronal systems, which are suitable for the analysis of Tau trafficking and sorting studies.
微管(MT)动力学研究对于了解细胞运输、细胞极性、轴突形成和其他神经发育机制至关重要。所有这些过程都依赖于微管蛋白聚合物在组装和拆卸之间的不断转换,即动态不稳定性。这一过程受到包括 Tau 蛋白在内的微管相关蛋白(MAP)磷酸化等因素的良好调控。蛋白激酶,尤其是微管亲和力调节激酶(MARK),可调节 MT-Tau 的相互作用,通过磷酸化诱导 Tau 解离。磷酸化的 Tau 从微管解离,形成不溶性的聚集体,即神经纤维缠结。这些高磷酸化 Tau 在神经元中的积聚破坏了细胞内基于 MT 的生理性运输机制,有可能导致神经退行性疾病的发生,如阿尔茨海默病(AD)和相关的 Tau 病。对MT细胞骨架动态的进一步研究至关重要,因为它们可以阐明神经退行性疾病(尤其是tau病)的病理机制以及基本的神经发育过程。MT网络的动态组装和解体研究需要活细胞成像,而不是基于固定样本的传统免疫细胞化学。为了研究MT的动态,我们对转染了荧光标记的微管加端追踪蛋白(+TIP)EB3的神经元进行了活细胞成像。这种蛋白与 MT 的生长末端结合,因此能实时观察到 MT 的生长。我们的成像分析方案可利用基于 ImageJ 的跟踪软件和肌动图确定转染神经元的体节和神经元中 MT 生长的数量、方向和速度。此外,在进行活体成像实验之前,还可以通过过表达或下调相应蛋白的实验来评估 Tau 和 MARK 激酶对 MT 细胞骨架的功能影响。我们使用了两种不同的人类神经元细胞模型:幼稚和分化的SH-SY5Y神经母细胞瘤细胞,以及诱导多能干细胞(iPSCs)衍生的神经元,这两种细胞都已成功地作为研究Tau相关病理的模型。本方案介绍了一种使用荧光标记的EB3蛋白作为微管加端标记物分析微管动态的优化方法。在本章中,我们概述了神经元转染、活细胞成像和基于 ImageJ 的必要延时图像分析的过程,这两个人源神经元系统适用于 Tau 转运和分选研究分析。
{"title":"Studying Microtubule Dynamics in Human Neurons: Two-Dimensional Microtubule Tracing and Kymographs in iPSC- and SH-SY5Y-Derived Neurons for Tau Research.","authors":"Nadine Allroggen, Helen Breuer, Sarah Bachmann, Michael Bell, Hans Zempel","doi":"10.1007/978-1-0716-3629-9_33","DOIUrl":"10.1007/978-1-0716-3629-9_33","url":null,"abstract":"<p><p>The study of microtubule (MT) dynamics is essential for the understanding of cellular transport, cell polarity, axon formation, and other neurodevelopmental mechanisms. All these processes rely on the constant transition between assembly and disassembly of tubulin polymers to/from MTs, known as dynamic instability. This process is well-regulated, among others, by phosphorylation of microtubule-associated proteins (MAP), including the Tau protein. Protein kinases, in particular the microtubule affinity regulating kinase (MARK), regulate the MT-Tau interaction, inducing Tau dissociation by phosphorylation. Phosphorylated Tau dissociates from microtubules forming insoluble aggregates known as neurofibrillary tangles. These accumulations of hyperphosphorylated Tau in the neurons disrupt the physiological MT-based transport machinery within the cell and can potentially lead to the development of neurodegenerative disorders, such as Alzheimer's disease (AD) and related tauopathies. Further investigations on the MT cytoskeleton dynamics are essential as they may elucidate pathomechanisms of neurodegenerative diseases - particularly tauopathies - as well as fundamental neurodevelopmental processes.The study of the dynamic assembly and disassembly of the MT network requires live-cell imaging rather than conventional immunocytochemistry based on fixed samples. To investigate MT dynamics, we perform live-cell imaging of neurons transfected with a fluorescently tagged version of the microtubule plus-end tracking protein (+TIP) EB3. This protein associates with the growing ends of MTs and thus visualizes MT growth in real time. Our imaging analysis protocol allows the determination of quantity, orientation, and velocity of MT growth in the soma and neurites of transfected neurons, using ImageJ-based tracking software and kymographs. Furthermore, functional effects of Tau and MARK kinases on the MT cytoskeleton can be assessed by overexpression or downregulation experiments of the respective protein prior to the live imaging assay. We use two different human neuronal cell models, naive and differentiated SH-SY5Y neuroblastoma cells, and neurons derived from induced pluripotent stem cells (iPSCs), both of which have shown success as models to study Tau-related pathologies.This protocol describes an optimized method for analysis of microtubule dynamics using fluorescent tagged EB3 protein as microtubule plus end marker. In this chapter, we outline the process of neuronal transfection, live-cell imaging, and necessary time-lapse image analysis based on ImageJ in two human-derived neuronal systems, which are suitable for the analysis of Tau trafficking and sorting studies.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2754 ","pages":"561-580"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140184798","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-01DOI: 10.1007/978-1-0716-3629-9_9
Urmi Sengupta, Rakez Kayed
Tau oligomers have been shown to be the main toxic tau species in several neurodegenerative disorders. To study tau oligomers, we have developed reagents and established methods for the reliable preparation, isolation, and detection of tau oligomers as well as their seeding and propagation both in vitro and in vivo. Detailed below are methods for isolation of tau oligomers from brain tissues and detection of tau oligomers using tau oligomer-specific antibodies by biochemical, immunohistochemical, and biophysical methods. Further, methods for evaluating the biological activity of the tau oligomers including their effects on synaptic function, seeding, and propagation in cell models and in vivo are also described.
Tau 寡聚体已被证明是多种神经退行性疾病中主要的毒性 tau 种类。为了研究 tau 低聚物,我们开发了试剂并建立了可靠的方法,用于制备、分离和检测 tau 低聚物,以及在体外和体内播种和繁殖。下面详细介绍从脑组织中分离 tau 低聚物的方法,以及通过生化、免疫组化和生物物理方法使用 tau 低聚物特异性抗体检测 tau 低聚物的方法。此外,还介绍了评估 tau 低聚物生物活性的方法,包括其对细胞模型和体内突触功能、播种和传播的影响。
{"title":"Tau Oligomers as Pathogenic Seeds: Preparation, Characterization, and Propagation In Vitro and In Vivo.","authors":"Urmi Sengupta, Rakez Kayed","doi":"10.1007/978-1-0716-3629-9_9","DOIUrl":"10.1007/978-1-0716-3629-9_9","url":null,"abstract":"<p><p>Tau oligomers have been shown to be the main toxic tau species in several neurodegenerative disorders. To study tau oligomers, we have developed reagents and established methods for the reliable preparation, isolation, and detection of tau oligomers as well as their seeding and propagation both in vitro and in vivo. Detailed below are methods for isolation of tau oligomers from brain tissues and detection of tau oligomers using tau oligomer-specific antibodies by biochemical, immunohistochemical, and biophysical methods. Further, methods for evaluating the biological activity of the tau oligomers including their effects on synaptic function, seeding, and propagation in cell models and in vivo are also described.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2754 ","pages":"147-183"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140184800","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-01DOI: 10.1007/978-1-0716-3738-8_13
Xiaoshan Shi, Gisele V Baracho, Woodrow E Lomas, Hye-Won Song, Stephanie J Widmann, Aaron J Tyznik
The paired detection of the transcriptome and proteome at single-cell resolution provides exquisite insight to immune mechanisms in health and disease. Here, we describe a detailed protocol wherein we combine cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), a technique utilizing antibody-derived tags (ADTs) to profile mRNA and proteins simultaneously via sequencing, with fluorescence-activated cell sorting to enrich cell populations. Our protocol provides step-by-step guidance on co-staining cells with both fluorescent antibodies and ADTs simultaneously, instructions on cell sorting and an overview of the single-cell capture workflow using the BD Rhapsody™ system. This method is useful for in-depth single-cell characterization on sorted rare cell populations.
{"title":"Co-staining with Fluorescent Antibodies and Antibody-Derived Tags for Cell Sorting Prior to CITE-Seq.","authors":"Xiaoshan Shi, Gisele V Baracho, Woodrow E Lomas, Hye-Won Song, Stephanie J Widmann, Aaron J Tyznik","doi":"10.1007/978-1-0716-3738-8_13","DOIUrl":"10.1007/978-1-0716-3738-8_13","url":null,"abstract":"<p><p>The paired detection of the transcriptome and proteome at single-cell resolution provides exquisite insight to immune mechanisms in health and disease. Here, we describe a detailed protocol wherein we combine cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), a technique utilizing antibody-derived tags (ADTs) to profile mRNA and proteins simultaneously via sequencing, with fluorescence-activated cell sorting to enrich cell populations. Our protocol provides step-by-step guidance on co-staining cells with both fluorescent antibodies and ADTs simultaneously, instructions on cell sorting and an overview of the single-cell capture workflow using the BD Rhapsody™ system. This method is useful for in-depth single-cell characterization on sorted rare cell populations.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2779 ","pages":"287-303"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140288489","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}