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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. 新型胎牛血清(FBS)及其冻干形式在 ELISpot 和 FluoroSpot 检测中的性能和稳定性:应用于临床试验中监测疫苗、细胞和基因免疫疗法的免疫反应。
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 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.

干扰素-γ(IFNγ)ELISpot 和 FluoroSpot 是免疫疗法临床研究中广泛使用的检测功能性细胞反应的化验方法。这些检测方法因其在疫苗开发研究中量化免疫反应的重要性而得到认可,最近又在细胞和基因治疗以及癌症免疫治疗领域崭露头角,因为在这些领域中,针对癌症新抗原的反应不容易在检测背景之上被检测到。在这里,我们测试了一种新型胎牛血清(FBS)--CultraPure FBS 在体外 ELISpot 和 FluoroSpot 检测以及体外扩增后的培养 FluoroSpot 检测中的应用。我们将几批通过冻干过程特别配制的 CultraPure FBS(冻干 FBS)与液体 CultraPure FBS 进行了比较。我们用稀释在补充了液态 CultraPure FBS 或冻干 FBS 的培养基中的抗原特异性肽池刺激人 PBMC,结果发现液态和冻干 FBS 产生的细胞因子数量相当,检测背景几乎可以忽略不计。此外,冻干 FBS 在体内外直接试验和体外培养试验中均表现出批次间的一致性和 90 天冷藏(4 °C)稳定性。此外,我们在此介绍一种使用 lyo-FBS 扩增低频抗原特异性 T 细胞的方法,该方法通过培养 FluoroSpot 检测法模拟了癌症新抗原的低频特异性。我们的结果表明,使用添加了 lyo-FBS 的培养基培养 9 天后,抗原特异性多功能 T 细胞会产生 Granzyme B、γ 干扰素 (IFNγ) 和肿瘤坏死因子 (TNF)。
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引用次数: 0
Dissociation of Placental Tissues for Single-Cell Techniques. 为单细胞技术分离胎盘组织
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 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.

将整个组织解离成单细胞悬浮液是重点分析单个细胞的技术的关键步骤。在此,我们介绍了一种从人类胎盘组织(基底板(BP)、胎盘绒毛(PV)和绒毛膜(CAM))制备高质量单细胞悬浮液的方案。该方案还为单细胞悬液的冷冻保存和回收提供了指导,以便日后使用。这里介绍的方法已被证明适用于下游单细胞应用,如单细胞 RNA 测序,这些应用需要有活力的高质量细胞悬浮液。
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引用次数: 0
Feto-Maternal Interface Organ-on-Chip: A New Technology to Study Ascending Infection. 胎儿-母体界面片上器官:研究上升感染的新技术
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 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.

人类妊娠建模具有挑战性,因为必须同时评估母亲和胎儿这两个主体。为了了解妊娠、分娩和不良妊娠结局,需要对妊娠期间形成的两个胎-母界面(即胎盘和胎膜)进行研究,以了解它们的生物学作用,器官功能障碍可导致不良妊娠结局。胎膜早破、自发性早产、子痫前期、宫内生长受限和妊娠糖尿病等不良妊娠结局在全球范围内呈上升趋势,这凸显了未来研究的必要性,以及更好地了解导致疾病发生的分子和细胞途径的必要性。目前的体内动物模型和体外细胞培养系统都无法回答这些问题,因为它们不能模拟人类调频干扰素的功能或结构。微流控芯片器官(OOC)装置利用微加工和软光刻技术,在小型体外平台上模拟复杂器官和器官系统的解剖结构和生物功能。FMi-OOC是一个四细胞培养室,同心圆系统,包含胎儿(羊膜)和母体(蜕膜)细胞层,已被验证可模拟妊娠的生理和病理状态(即上升感染、全身氧化应激和母体毒物暴露)。该平台与显微镜和生化分析等各种分析方法完全兼容。本实验方案将概述该装置的制造、细胞装载以及用于模拟上升期感染相关不良妊娠结局的实用性。
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引用次数: 0
Rapid DNA-FISH in Arabidopsis thaliana Somatic Cells. 拟南芥体细胞中的快速 DNA-FISH。
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 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。
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引用次数: 0
Simultaneous In Situ Detection of m6A-Modified and Unmodified RNAs Using DART-FISH. 利用 DART-FISH 同时原位检测 m6A 修饰和未修饰的 RNA。
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 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。
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引用次数: 0
Cryo-electron Microscopy and Molecular Modeling Methods to Characterize the Dynamics of Tau Bound to Microtubules. 用冷冻电子显微镜和分子建模方法描述与微管结合的 Tau 的动力学特征
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 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.

电子显微镜元推理综合结构生物学方法能够将冷冻电子显微镜电子密度图与分子动力学等分子建模技术相结合,以高效的方式揭示原子论生物分子结构集合和图谱数据的误差。在此,我们展示了电子显微镜元参考方案和分析方法,通过使用最先进的分子力学力场和电子密度图,阐明了与微管结合的微管相关蛋白tau的原子结构组合。
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引用次数: 0
Identification of Tau Toxicity Modifiers in the Drosophila Eye. 果蝇眼睛中 Tau 毒性修饰因子的鉴定
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 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.

果蝇是研究人类疾病的强大模型,这得益于它的遗传工具和筛选简便性。人类基因可在目标器官中表达,其毒性可通过易于检测的外部表型进行评估,这些表型可用作毒性调节剂基因筛选的读数。在本章中,我将介绍如何在果蝇眼睛中表达人类Tau蛋白,通过Western印迹评估蛋白表达,通过量化Tau诱导的粗糙眼睛的大小评估Tau毒性,以及在果蝇眼睛中进行Tau毒性调节剂的基因筛选。
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引用次数: 0
Studying Microtubule Dynamics in Human Neurons: Two-Dimensional Microtubule Tracing and Kymographs in iPSC- and SH-SY5Y-Derived Neurons for Tau Research. 研究人类神经元中的微管动力学:用于 Tau 研究的 iPSC 和 SH-SY5Y 衍生神经元的二维微管追踪和 Kymographs。
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 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。
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引用次数: 0
Tau Oligomers as Pathogenic Seeds: Preparation, Characterization, and Propagation In Vitro and In Vivo. 作为致病种子的 Tau 寡聚体:体外和体内制备、表征和繁殖
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 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。
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引用次数: 0
Co-staining with Fluorescent Antibodies and Antibody-Derived Tags for Cell Sorting Prior to CITE-Seq. 在进行 CITE-Seq 分析之前,使用荧光抗体和抗体衍生标签进行细胞分选。
Q4 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-01 DOI: 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.

以单细胞分辨率对转录组和蛋白质组进行配对检测,可以深入了解健康和疾病中的免疫机制。在这里,我们描述了一个详细的方案,其中我们将通过测序对转录组和表位进行细胞索引(CITE-Seq)(一种利用抗体衍生标签(ADTs)通过测序同时分析 mRNA 和蛋白质的技术)与荧光激活细胞分拣相结合,以富集细胞群。我们的方案逐步指导如何同时用荧光抗体和 ADTs 对细胞进行共染,指导如何进行细胞分选,并概述了使用 BD Rhapsody™ 系统的单细胞捕获工作流程。这种方法适用于对分选的稀有细胞群进行深入的单细胞表征。
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引用次数: 0
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Methods in molecular biology
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