Jillian E J Cox, Kevin D Pham, Alex W Keck, Zsabre Wright, Manu A Thomas, Willard M Freeman, Sarah R Ocañas
Microglia, the brain's primary resident immune cell, exists in various phenotypic states depending on intrinsic and extrinsic signaling. Distinguishing between these phenotypes can offer valuable biological insights into neurodevelopmental and neurodegenerative processes. Recent advances in single-cell transcriptomic profiling have allowed for increased granularity and better separation of distinct microglial states. While techniques such as immunofluorescence and single-cell RNA sequencing (scRNA-seq) are available to differentiate microglial phenotypes and functions, these methods present notable limitations, including challenging quantification methods, high cost, and advanced analytical techniques. This protocol addresses these limitations by presenting an optimized cell preparation procedure that prevents ex vivo activation and a flow cytometry panel to distinguish four distinct microglial states from murine brain tissue. Following cell preparation, fluorescent antibodies were applied to label 1) homeostatic, 2) disease-associated (DAM), 3) interferon response (IRM), and 4) lipid-droplet accumulating (LDAM) microglia, based on gene markers identified in previous scRNA-Seq studies. Stained cells were analyzed by flow cytometry to assess phenotypic distribution as a function of age and sex. A key advantage of this procedure is its adaptability, allowing the panel provided to be enhanced using additional markers with an appropriate cell analyzer (i.e., Cytek Aurora 5 laser spectral flow cytometer) and interrogating different brain regions or disease models. Additionally, this protocol does not require microglial cell sorting, resulting in a relatively quick and straightforward experiment. Ultimately, this protocol can compare the distribution of microglial phenotypic states between various experimental groups, such as disease state or age, with a lower cost and higher throughput than scRNA-seq. Key features • Analysis of microglial phenotypes from murine brain without the need for cell sorting, imaging, or scRNA-seq. • This protocol can distinguish between homeostatic, disease-associated (DAM), lipid-droplet accumulating (LDAM), and interferon response (IRM) microglia from any murine brain region and/or disease model of interest. • This protocol can be modified to incorporate additional markers of interest or dyes when using a cell analyzer capable of multiple color detections.
{"title":"Flow Cytometry Analysis of Microglial Phenotypes in the Murine Brain During Aging and Disease.","authors":"Jillian E J Cox, Kevin D Pham, Alex W Keck, Zsabre Wright, Manu A Thomas, Willard M Freeman, Sarah R Ocañas","doi":"10.21769/BioProtoc.5018","DOIUrl":"10.21769/BioProtoc.5018","url":null,"abstract":"<p><p>Microglia, the brain's primary resident immune cell, exists in various phenotypic states depending on intrinsic and extrinsic signaling. Distinguishing between these phenotypes can offer valuable biological insights into neurodevelopmental and neurodegenerative processes. Recent advances in single-cell transcriptomic profiling have allowed for increased granularity and better separation of distinct microglial states. While techniques such as immunofluorescence and single-cell RNA sequencing (scRNA-seq) are available to differentiate microglial phenotypes and functions, these methods present notable limitations, including challenging quantification methods, high cost, and advanced analytical techniques. This protocol addresses these limitations by presenting an optimized cell preparation procedure that prevents ex vivo activation and a flow cytometry panel to distinguish four distinct microglial states from murine brain tissue. Following cell preparation, fluorescent antibodies were applied to label 1) homeostatic, 2) disease-associated (DAM), 3) interferon response (IRM), and 4) lipid-droplet accumulating (LDAM) microglia, based on gene markers identified in previous scRNA-Seq studies. Stained cells were analyzed by flow cytometry to assess phenotypic distribution as a function of age and sex. A key advantage of this procedure is its adaptability, allowing the panel provided to be enhanced using additional markers with an appropriate cell analyzer (i.e., Cytek Aurora 5 laser spectral flow cytometer) and interrogating different brain regions or disease models. Additionally, this protocol does not require microglial cell sorting, resulting in a relatively quick and straightforward experiment. Ultimately, this protocol can compare the distribution of microglial phenotypic states between various experimental groups, such as disease state or age, with a lower cost and higher throughput than scRNA-seq. Key features • Analysis of microglial phenotypes from murine brain without the need for cell sorting, imaging, or scRNA-seq. • This protocol can distinguish between homeostatic, disease-associated (DAM), lipid-droplet accumulating (LDAM), and interferon response (IRM) microglia from any murine brain region and/or disease model of interest. • This protocol can be modified to incorporate additional markers of interest or dyes when using a cell analyzer capable of multiple color detections.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11211077/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141473541","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The intricate composition, heterogeneity, and hierarchical organization of the human bone marrow hematopoietic microenvironment (HME) present challenges for experimentation, which is primarily due to the scarcity of HME-forming cells, notably bone marrow stromal cells (BMSCs). The limited understanding of non-hematopoietic cell phenotypes complicates the unraveling of the HME's intricacies and necessitates a precise isolation protocol for systematic studies. The protocol presented herein puts special emphasis on the accuracy and high quality of BMSCs obtained for downstream sequencing analysis. Utilizing CD45 and CD235a as negative markers ensures sufficient enrichment of non-hematopoietic cells within the HME. By adding positive selection based on CD271 expression, this protocol allows for selectively isolating the rare and pivotal bona fide stromal cell population with high precision. The outlined step-by-step protocol provides a robust tool for isolating and characterizing non-hematopoietic cells, including stromal cells, from human bone marrow preparations. This approach thus contributes valuable information to promote research in a field that is marked by a scarcity of studies and helps to conduct important experimentation that will deepen our understanding of the intricate cellular interactions within the bone marrow niche. Key features • Isolation of high-quality human non-hematopoietic bone marrow cells for scRNAseq • Targeted strategy for enriching low-frequency stromal cells.
{"title":"Isolation of Human Bone Marrow Non-hematopoietic Cells for Single-cell RNA Sequencing.","authors":"Hongzhe Li, Sandro Bräunig, Stefan Scheding","doi":"10.21769/BioProtoc.5020","DOIUrl":"10.21769/BioProtoc.5020","url":null,"abstract":"<p><p>The intricate composition, heterogeneity, and hierarchical organization of the human bone marrow hematopoietic microenvironment (HME) present challenges for experimentation, which is primarily due to the scarcity of HME-forming cells, notably bone marrow stromal cells (BMSCs). The limited understanding of non-hematopoietic cell phenotypes complicates the unraveling of the HME's intricacies and necessitates a precise isolation protocol for systematic studies. The protocol presented herein puts special emphasis on the accuracy and high quality of BMSCs obtained for downstream sequencing analysis. Utilizing CD45 and CD235a as negative markers ensures sufficient enrichment of non-hematopoietic cells within the HME. By adding positive selection based on CD271 expression, this protocol allows for selectively isolating the rare and pivotal <i>bona fide</i> stromal cell population with high precision. The outlined step-by-step protocol provides a robust tool for isolating and characterizing non-hematopoietic cells, including stromal cells, from human bone marrow preparations. This approach thus contributes valuable information to promote research in a field that is marked by a scarcity of studies and helps to conduct important experimentation that will deepen our understanding of the intricate cellular interactions within the bone marrow niche. Key features • Isolation of high-quality human non-hematopoietic bone marrow cells for scRNAseq • Targeted strategy for enriching low-frequency stromal cells.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11211075/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141473542","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
All aerial organs in plants originate from the shoot apical meristem, a specialized tissue at the tip of a plant, enclosing a few stem cells. Understanding developmental dynamics within this tissue in relation to internal and external stimuli is of crucial importance. Imaging the meristem at the cellular level beyond very early stages requires the apex to be detached from the plant body, a procedure that does not allow studies in living, intact plants over longer periods. This protocol describes a new confocal microscopy method with the potential to image the shoot apical meristem of an intact, soil-grown, flowering Arabidopsis plant over several days. The setup opens new avenues to study apical stem cells, their interconnection with the whole plant, and their responses to environmental stimuli. Key features • Novel dissection and imaging method of the shoot apical meristem of Arabidopsis. • Procedure performed with intact, soil-grown, flowering plants. • Possibility of long-term live imaging of the shoot apical meristem. • Protocol can be adapted to different plant species.
{"title":"Live Imaging of the Shoot Apical Meristem of Intact, Soil-Grown, Flowering <i>Arabidopsis</i> Plants.","authors":"Gabriele Bradamante","doi":"10.21769/BioProtoc.5015","DOIUrl":"10.21769/BioProtoc.5015","url":null,"abstract":"<p><p>All aerial organs in plants originate from the shoot apical meristem, a specialized tissue at the tip of a plant, enclosing a few stem cells. Understanding developmental dynamics within this tissue in relation to internal and external stimuli is of crucial importance. Imaging the meristem at the cellular level beyond very early stages requires the apex to be detached from the plant body, a procedure that does not allow studies in living, intact plants over longer periods. This protocol describes a new confocal microscopy method with the potential to image the shoot apical meristem of an intact, soil-grown, flowering Arabidopsis plant over several days. The setup opens new avenues to study apical stem cells, their interconnection with the whole plant, and their responses to environmental stimuli. Key features • Novel dissection and imaging method of the shoot apical meristem of <i>Arabidopsis</i>. • Procedure performed with intact, soil-grown, flowering plants. • Possibility of long-term live imaging of the shoot apical meristem. • Protocol can be adapted to different plant species.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11211079/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141473543","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The Auxin-inducible degron (AID) system is a genetic tool that induces rapid target protein depletion in an auxin-dependent manner. Recently, two advanced AID systems-the super-sensitive AID and AID 2-were developed using an improved pair of synthetic auxins and mutated TIR1 proteins. In these AID systems, a nanomolar concentration of synthetic auxins is sufficient as a degradation inducer for target proteins. However, despite these advancements, AID systems still require the fusion of an AID tag to the target protein for degradation, potentially affecting its function and stability. To address this limitation, we developed an affinity linker-based super-sensitive AID (AlissAID) system using a single peptide antibody known as a nanobody. In this system, the degradation of GFP- or mCherry-tagged target proteins is induced in a synthetic auxin (5-Ad-IAA)-dependent manner. Here, we introduce a simple method for generating AlissAID strains targeting GFP or mCherry fusion proteins in budding yeasts. Key features • AlissAID system enables efficient degradation of the GFP or mCherry fusion proteins in a 5-Ad-IAA-depending manner. • Transforming the pAlissAID plasmids into strains with GFP- or mCherry- tagged proteins.
辅助素诱导降解子(AID)系统是一种遗传工具,能以辅助素依赖的方式诱导目标蛋白的快速消耗。最近,利用一对改进的合成辅助素和突变的 TIR1 蛋白,开发出了两种先进的 AID 系统--超灵敏 AID 和 AID 2。在这些 AID 系统中,纳摩尔浓度的合成助剂就足以诱导目标蛋白质降解。然而,尽管取得了这些进步,AID 系统仍然需要将 AID 标签与目标蛋白融合才能降解,这可能会影响目标蛋白的功能和稳定性。为了解决这一局限性,我们开发了一种基于亲和连接体的超灵敏 AID(AlissAID)系统,使用的是一种称为纳米抗体的单肽抗体。在该系统中,GFP 或 mCherry 标记的目标蛋白的降解是以合成辅助素(5-Ad-IAA)依赖的方式诱导的。在这里,我们介绍了一种在芽殖酵母中生成靶向 GFP 或 mCherry 融合蛋白的 AlissAID 菌株的简单方法。主要特点 - AlissAID 系统能以 5-Ad-IAA 依赖性方式高效降解 GFP 或 mCherry 融合蛋白。- 将 pAlissAID 质粒转化为带有 GFP 或 mCherry 标记蛋白的菌株。
{"title":"A Single-step Generation of AlissAID-based Conditional Knockdown Strains Using Nanobody that Targets GFP or mCherry in Budding Yeast.","authors":"Yoshitaka Ogawa, Taisei P Ueda, Keisuke Obara, Kohei Nishimura, Takumi Kamura","doi":"10.21769/BioProtoc.5019","DOIUrl":"10.21769/BioProtoc.5019","url":null,"abstract":"<p><p>The Auxin-inducible degron (AID) system is a genetic tool that induces rapid target protein depletion in an auxin-dependent manner. Recently, two advanced AID systems-the super-sensitive AID and AID 2-were developed using an improved pair of synthetic auxins and mutated TIR1 proteins. In these AID systems, a nanomolar concentration of synthetic auxins is sufficient as a degradation inducer for target proteins. However, despite these advancements, AID systems still require the fusion of an AID tag to the target protein for degradation, potentially affecting its function and stability. To address this limitation, we developed an affinity linker-based super-sensitive AID (AlissAID) system using a single peptide antibody known as a nanobody. In this system, the degradation of GFP- or mCherry-tagged target proteins is induced in a synthetic auxin (5-Ad-IAA)-dependent manner. Here, we introduce a simple method for generating AlissAID strains targeting GFP or mCherry fusion proteins in budding yeasts. Key features • AlissAID system enables efficient degradation of the GFP or mCherry fusion proteins in a 5-Ad-IAA-depending manner. • Transforming the pAlissAID plasmids into strains with GFP- or mCherry- tagged proteins.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11211076/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141473539","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sen Wang, Jianyu Wang, Dongfang Li, Fangwei Chen, Wanxin Luo, Junlong Zhao, Lan He
Human babesiosis is a tick-borne disease caused by Babesia pathogens. The disease, which presents with malaria-like symptoms, can be life-threatening, especially in individuals with weakened immune systems and the elderly. The worldwide prevalence of human babesiosis has been gradually rising, prompting alarm among public health experts. In other pathogens, genetic techniques have proven to be valuable tools for conducting functional studies to understand the importance of specific genes in development and pathogenesis as well as to validate novel cellular targets for drug discovery. Genetic manipulation methods have been established for several non-human Babesia and Theileria species and, more recently, have begun to be developed for human Babesia parasites. We have previously reported the development of a method for genetic manipulation of the human pathogen Babesia duncani. This method is based on positive selection using the hDHFR gene as a selectable marker, whose expression is regulated by the ef-1aB promoter, along with homology regions that facilitate integration into the gene of interest through homologous recombination. Herein, we provide a detailed description of the steps needed to implement this strategy in B. duncani to study gene function. It is anticipated that the implementation of this method will significantly improve our understanding of babesiosis and facilitate the development of novel and more effective therapeutic strategies for the treatment of human babesiosis. Key features This protocol provides an effective means of transfection of B. duncani, enabling genetic manipulation and editing to gain further insights into its biology and pathogenesis. The protocol outlined here for the electroporation of B. duncani represents an advancement over previous methods used for B. bovis [1]. Improvements include higher volume of culture used during the electroporation step and an enhancement in the number of electroporation pulses. These modifications likely enhance the efficiency of gene editing in B. duncani, allowing for quicker and more effective selection of transgenic parasites.
人类巴贝西亚原虫病是一种由巴贝西亚原虫病原体引起的蜱媒疾病。这种疾病会出现类似疟疾的症状,可危及生命,尤其是免疫力低下的人和老年人。人类巴贝西亚原虫病在全球的发病率逐渐上升,引起了公共卫生专家的警觉。在其他病原体中,基因技术已被证明是进行功能研究的重要工具,可用于了解特定基因在发育和致病过程中的重要性,以及验证用于药物发现的新型细胞靶点。针对几种非人类巴贝西亚原虫和蒂勒氏原虫的基因操作方法已经建立,最近又开始针对人类巴贝西亚原虫寄生虫开发基因操作方法。我们曾报道过一种对人类病原体巴贝西亚登卡尼虫进行基因操纵的方法。这种方法的基础是使用 hDHFR 基因作为可选择标记进行正选择,该基因的表达受 ef-1aB 启动子以及同源区的调控,这些同源区有利于通过同源重组整合到感兴趣的基因中。在此,我们将详细介绍在 B. duncani 中实施这一策略以研究基因功能所需的步骤。预计该方法的实施将极大地提高我们对巴贝西亚原虫病的认识,并有助于开发新的、更有效的治疗策略来治疗人类巴贝西亚原虫病。主要特点 本方案提供了转染巴贝西亚原虫的有效方法,可进行基因操作和编辑,从而进一步了解其生物学和致病机理。这里概述的邓卡尼巴贝母电穿孔方案比以前用于牛巴贝母的方法更先进[1]。改进之处包括在电穿孔步骤中使用了更多的培养物,并增加了电穿孔脉冲的次数。这些改进可能会提高 B. duncani 基因编辑的效率,从而更快、更有效地筛选出转基因寄生虫。
{"title":"Transfection of <i>Babesia duncani</i>: A Genetic Toolbox of this Pathogen to Advance <i>Babesia</i> Biology.","authors":"Sen Wang, Jianyu Wang, Dongfang Li, Fangwei Chen, Wanxin Luo, Junlong Zhao, Lan He","doi":"10.21769/BioProtoc.5016","DOIUrl":"10.21769/BioProtoc.5016","url":null,"abstract":"<p><p>Human babesiosis is a tick-borne disease caused by <i>Babesia</i> pathogens. The disease, which presents with malaria-like symptoms, can be life-threatening, especially in individuals with weakened immune systems and the elderly. The worldwide prevalence of human babesiosis has been gradually rising, prompting alarm among public health experts. In other pathogens, genetic techniques have proven to be valuable tools for conducting functional studies to understand the importance of specific genes in development and pathogenesis as well as to validate novel cellular targets for drug discovery. Genetic manipulation methods have been established for several non-human <i>Babesia</i> and <i>Theileria</i> species and, more recently, have begun to be developed for human Babesia parasites. We have previously reported the development of a method for genetic manipulation of the human pathogen <i>Babesia duncani</i>. This method is based on positive selection using the hDHFR gene as a selectable marker, whose expression is regulated by the ef-1aB promoter, along with homology regions that facilitate integration into the gene of interest through homologous recombination. Herein, we provide a detailed description of the steps needed to implement this strategy in <i>B. duncani</i> to study gene function. It is anticipated that the implementation of this method will significantly improve our understanding of babesiosis and facilitate the development of novel and more effective therapeutic strategies for the treatment of human babesiosis. Key features This protocol provides an effective means of transfection of <i>B. duncani</i>, enabling genetic manipulation and editing to gain further insights into its biology and pathogenesis. The protocol outlined here for the electroporation of <i>B. duncani</i> represents an advancement over previous methods used for <i>B. bovis</i> [1]. Improvements include higher volume of culture used during the electroporation step and an enhancement in the number of electroporation pulses. These modifications likely enhance the efficiency of gene editing in <i>B. duncani</i>, allowing for quicker and more effective selection of transgenic parasites.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11211078/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141473545","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Alex Shepherd, Bigitha Bennychen, Zafer Ahmed, Risini D Weeratna, Scott McComb
Chimeric antigen receptors (CARs) are synthetic fusion proteins that can reprogram immune cells to target specific antigens. CAR-expressing T cells have emerged as an effective treatment method for hematological cancers; despite this success, the mechanisms and structural properties that govern CAR responses are not fully understood. Here, we provide a simple assay to assess cellular avidity using a standard flow cytometer. This assay measures the interaction kinetics of CAR-expressing T cells and targets antigen-expressing target cells. By co-culturing stably transfected CAR Jurkat cells with target positive and negative cells for short periods of time in a varying effector-target gradient, we were able to observe the formation of CAR-target cell doublets, providing a readout of actively bound cells. When using the optimized protocol reported here, we observed unique cellular binding curves that varied between CAR constructs with differing antigen binding domains. The cellular binding kinetics of unique CARs remained consistent, were dependent on specific target antigen expression, and required active biological signaling. While existing literature is not clear at this time whether higher or lower CAR cell binding is beneficial to CAR therapeutic activity, the application of this simplified protocol for assessing CAR binding could lead to a better understanding of the proximal signaling events that regulate CAR functionality. Key features • Determines CAR receptor cellular interaction kinetics using a Jurkat cell model. • Can be used for a wide variety of CAR target antigens, including both hematological and solid tumor targets. • Experiments can be performed in under two hours with no staining using a standard flow cytometer. • Requires stable CAR Jurkat cells and target cells with stable fluorescent marker expression for optimal results.
嵌合抗原受体(CAR)是一种人工合成的融合蛋白,可以重新编程免疫细胞,使其针对特定抗原。表达 CAR 的 T 细胞已成为治疗血液肿瘤的一种有效方法;尽管取得了这一成功,但人们对影响 CAR 反应的机制和结构特性还不完全了解。在此,我们提供了一种简单的检测方法,利用标准流式细胞仪评估细胞的亲和性。这种检测方法可测量表达 CAR 的 T 细胞与靶抗原表达靶细胞的相互作用动力学。通过将稳定转染的 CAR Jurkat 细胞与靶标阳性细胞和阴性细胞在不同的效应物-靶标梯度中短时间共培养,我们能够观察到 CAR-靶标细胞双倍体的形成,从而提供主动结合细胞的读数。在使用本文报告的优化方案时,我们观察到了独特的细胞结合曲线,这些曲线在具有不同抗原结合域的 CAR 构建物之间各不相同。独特 CAR 的细胞结合动力学保持一致,取决于特定靶抗原的表达,并且需要活跃的生物信号传导。虽然现有文献尚不清楚较高或较低的 CAR 细胞结合力是否有利于 CAR 的治疗活性,但应用这种简化的 CAR 结合力评估方案可以更好地了解调控 CAR 功能的近端信号事件。主要特点 - 使用 Jurkat 细胞模型确定 CAR 受体细胞相互作用动力学。- 可用于多种 CAR 靶抗原,包括血液和实体瘤靶点。- 使用标准流式细胞仪可在两小时内完成实验,无需染色。- 需要稳定的 CAR Jurkat 细胞和具有稳定荧光标记表达的靶细胞,才能获得最佳结果。
{"title":"A Flow Cytometry-Based Method for Assessing CAR Cell Binding Kinetics Using Stable CAR Jurkat Cells.","authors":"Alex Shepherd, Bigitha Bennychen, Zafer Ahmed, Risini D Weeratna, Scott McComb","doi":"10.21769/BioProtoc.5021","DOIUrl":"10.21769/BioProtoc.5021","url":null,"abstract":"<p><p>Chimeric antigen receptors (CARs) are synthetic fusion proteins that can reprogram immune cells to target specific antigens. CAR-expressing T cells have emerged as an effective treatment method for hematological cancers; despite this success, the mechanisms and structural properties that govern CAR responses are not fully understood. Here, we provide a simple assay to assess cellular avidity using a standard flow cytometer. This assay measures the interaction kinetics of CAR-expressing T cells and targets antigen-expressing target cells. By co-culturing stably transfected CAR Jurkat cells with target positive and negative cells for short periods of time in a varying effector-target gradient, we were able to observe the formation of CAR-target cell doublets, providing a readout of actively bound cells. When using the optimized protocol reported here, we observed unique cellular binding curves that varied between CAR constructs with differing antigen binding domains. The cellular binding kinetics of unique CARs remained consistent, were dependent on specific target antigen expression, and required active biological signaling. While existing literature is not clear at this time whether higher or lower CAR cell binding is beneficial to CAR therapeutic activity, the application of this simplified protocol for assessing CAR binding could lead to a better understanding of the proximal signaling events that regulate CAR functionality. Key features • Determines CAR receptor cellular interaction kinetics using a Jurkat cell model. • Can be used for a wide variety of CAR target antigens, including both hematological and solid tumor targets. • Experiments can be performed in under two hours with no staining using a standard flow cytometer. • Requires stable CAR Jurkat cells and target cells with stable fluorescent marker expression for optimal results.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11211074/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141473579","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Lizhe Xu, Amy Berninger, Steven M Lakin, Vivian O'Donnell, Jim L Pierce, Steven J Pauszek, Roger W Barrette, Bonto Faburay
Foot-and-mouth disease (FMD) is a severe and extremely contagious viral disease of cloven-hoofed domestic and wild animals, which leads to serious economic losses to the livestock industry globally. FMD is caused by the FMD virus (FMDV), a positive-strand RNA virus that belongs to the genus Aphthovirus, within the family Picornaviridae. Early detection and characterization of FMDV strains are key factors to control new outbreaks and prevent the spread of the disease. Here, we describe a direct RNA sequencing method using Oxford Nanopore Technology (ONT) Flongle flow cells on MinION Mk1C (or GridION) to characterize FMDV. This is a rapid, low cost, and easily deployed point of care (POC) method for a near real-time characterization of FMDV in endemic areas or outbreak investigation sites. Key features • Saves ~35 min of the original protocol time by omitting the reverse transcription step and lowers the costs of reagents and consumables. • Replaces the GridION flow cell from the original protocol with the Flongle, which saves ~90% on the flow cell cost. • Combines the NGS benchwork with a modified version of our African swine fever virus (ASFV) fast analysis pipeline to achieve FMDV characterization within minutes. Graphical overview Schematic of direct RNA sequencing of foot-and-mouth disease virus (FMDV) process, which takes ~50 min from extracted RNA to final loading, modified from the ONT SQK-RNA002 protocol (Version: DRS_9080_v2_revO_14Aug2019).
{"title":"Direct RNA Sequencing of Foot-and-mouth Disease Virus Genome Using a Flongle on MinION.","authors":"Lizhe Xu, Amy Berninger, Steven M Lakin, Vivian O'Donnell, Jim L Pierce, Steven J Pauszek, Roger W Barrette, Bonto Faburay","doi":"10.21769/BioProtoc.5017","DOIUrl":"10.21769/BioProtoc.5017","url":null,"abstract":"<p><p><i>Foot-and-mouth disease (FMD) is a severe and extremely contagious viral disease of cloven-hoofed domestic and wild animals, which leads to serious economic losses to the livestock industry globally. FMD is caused by the FMD virus (FMDV), a positive-strand RNA virus that belongs to the genus</i> Aphthovirus, within the family Picornaviridae. Early detection and characterization of FMDV strains are key factors to control new outbreaks and prevent the spread of the disease. Here, we describe a direct RNA sequencing method using Oxford Nanopore Technology (ONT) Flongle flow cells on MinION Mk1C (or GridION) to characterize FMDV. This is a rapid, low cost, and easily deployed point of care (POC) method for a near real-time characterization of FMDV in endemic areas or outbreak investigation sites. Key features • Saves ~35 min of the original protocol time by omitting the reverse transcription step and lowers the costs of reagents and consumables. • Replaces the GridION flow cell from the original protocol with the Flongle, which saves ~90% on the flow cell cost. • Combines the NGS benchwork with a modified version of our African swine fever virus (ASFV) fast analysis pipeline to achieve FMDV characterization within minutes. Graphical overview Schematic of direct RNA sequencing of foot-and-mouth disease virus (FMDV) process, which takes ~50 min from extracted RNA to final loading, modified from the ONT SQK-RNA002 protocol (Version: DRS_9080_v2_revO_14Aug2019).</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11211080/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141473540","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Fusun Doldur-Balli, Amber J Zimmerman, Christoph Seiler, Olivia Veatch, Allan I Pack
Sleep is an essential behavior that is still poorly understood. Sleep abnormalities accompany a variety of psychiatric and neurological disorders, and sleep can serve as a modifiable behavior in the treatment of these disorders. Zebrafish (Danio rerio) has proven to be a powerful model organism to study sleep and the interplay between sleep and these disorders due to the high conservation of the neuro-modulatory mechanisms that control sleep and wake states between zebrafish and humans. The zebrafish is a diurnal vertebrate with a relatively simple nervous system compared to mammalian models, exhibiting conservation of sleep ontogeny across different life stages. Zebrafish larvae are an established high-throughput model to assess sleep phenotypes and the biological underpinnings of sleep disturbances. To date, sleep measurement in juvenile and adult zebrafish has not been performed in a standardized and reproducible manner because of the relatively low-throughput nature in relation to their larval counterparts. This has left a gap in understanding sleep across later stages of life that are relevant to many psychiatric and neurodegenerative disorders. Several research groups have used homemade systems to address this gap. Here, we report employing commercially available equipment to track activity and sleep/wake patterns in juvenile and adult zebrafish. The equipment allows researchers to perform automated behavior assays in an isolated environment with light/dark and temperature control for multiple days. We first explain the experimental procedure to track the sleep and activity of adult zebrafish and then validate the protocol by measuring the effects of melatonin and DMSO administration. Key features • Allows an isolated and controllable environment to carry out activity and sleep assays in juvenile and adult zebrafish. • Measures activity of zebrafish in life stages later than early development, which requires feeding animals during the assay. • Requires use of a commercially available equipment system and six tanks. • The activity of zebrafish can be tracked for five days including an acclimation step.
{"title":"Measuring Sleep and Activity Patterns in Adult Zebrafish.","authors":"Fusun Doldur-Balli, Amber J Zimmerman, Christoph Seiler, Olivia Veatch, Allan I Pack","doi":"10.21769/BioProtoc.5014","DOIUrl":"10.21769/BioProtoc.5014","url":null,"abstract":"<p><p>Sleep is an essential behavior that is still poorly understood. Sleep abnormalities accompany a variety of psychiatric and neurological disorders, and sleep can serve as a modifiable behavior in the treatment of these disorders. Zebrafish (<i>Danio rerio</i>) has proven to be a powerful model organism to study sleep and the interplay between sleep and these disorders due to the high conservation of the neuro-modulatory mechanisms that control sleep and wake states between zebrafish and humans. The zebrafish is a diurnal vertebrate with a relatively simple nervous system compared to mammalian models, exhibiting conservation of sleep ontogeny across different life stages. Zebrafish larvae are an established high-throughput model to assess sleep phenotypes and the biological underpinnings of sleep disturbances. To date, sleep measurement in juvenile and adult zebrafish has not been performed in a standardized and reproducible manner because of the relatively low-throughput nature in relation to their larval counterparts. This has left a gap in understanding sleep across later stages of life that are relevant to many psychiatric and neurodegenerative disorders. Several research groups have used homemade systems to address this gap. Here, we report employing commercially available equipment to track activity and sleep/wake patterns in juvenile and adult zebrafish. The equipment allows researchers to perform automated behavior assays in an isolated environment with light/dark and temperature control for multiple days. We first explain the experimental procedure to track the sleep and activity of adult zebrafish and then validate the protocol by measuring the effects of melatonin and DMSO administration. Key features • Allows an isolated and controllable environment to carry out activity and sleep assays in juvenile and adult zebrafish. • Measures activity of zebrafish in life stages later than early development, which requires feeding animals during the assay. • Requires use of a commercially available equipment system and six tanks. • The activity of zebrafish can be tracked for five days including an acclimation step.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11211082/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141473544","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Extracellular vesicles (EVs) are a heterogeneous group of nanoparticles possessing a lipid bilayer membrane that plays a significant role in intercellular communication by transferring their cargoes, consisting of peptides, proteins, fatty acids, DNA, and RNA, to receiver cells. Isolation of EVs is cumbersome and time-consuming due to their nano size and the co-isolation of small molecules along with EVs. This is why current protocols for the isolation of EVs are unable to provide high purity. So far, studies have focused on EVs derived from cell supernatants or body fluids but are associated with a number of limitations. Cell lines with a high passage number cannot be considered as representative of the original cell type, and EVs isolated from those can present distinct properties and characteristics. Additionally, cultured cells only have a single cell type and do not possess any cellular interactions with other types of cells, which normally exist in the tissue microenvironment. Therefore, studies involving the direct EVs isolation from whole tissues can provide a better understanding of intercellular communication in vivo. This underscores the critical need to standardize and optimize protocols for isolating and characterizing EVs from tissues. We have developed a differential centrifugation-based technique to isolate and characterize EVs from whole adipose tissue, which can be potentially applied to other types of tissues. This may help us to better understand the role of EVs in the tissue microenvironment in both diseased and normal conditions. Key features • Isolation of tissue-derived extracellular vesicles from ex vivo culture of visceral adipose tissue or any whole tissue. • Microscopic visualization of extracellular vesicles' morphology without dehydration steps, with minimum effect on their shape. • Flow cytometry approach to characterize the extracellular vesicles using specific protein markers, as an alternative to the time-consuming western blot.
{"title":"Isolation and Characterization of Extracellular Vesicles Derived from Ex Vivo Culture of Visceral Adipose Tissue.","authors":"Ankita Arora, Vinit Sharma, Rajesh Gupta, Anjali Aggarwal","doi":"10.21769/BioProtoc.5011","DOIUrl":"10.21769/BioProtoc.5011","url":null,"abstract":"<p><p>Extracellular vesicles (EVs) are a heterogeneous group of nanoparticles possessing a lipid bilayer membrane that plays a significant role in intercellular communication by transferring their cargoes, consisting of peptides, proteins, fatty acids, DNA, and RNA, to receiver cells. Isolation of EVs is cumbersome and time-consuming due to their nano size and the co-isolation of small molecules along with EVs. This is why current protocols for the isolation of EVs are unable to provide high purity. So far, studies have focused on EVs derived from cell supernatants or body fluids but are associated with a number of limitations. Cell lines with a high passage number cannot be considered as representative of the original cell type, and EVs isolated from those can present distinct properties and characteristics. Additionally, cultured cells only have a single cell type and do not possess any cellular interactions with other types of cells, which normally exist in the tissue microenvironment. Therefore, studies involving the direct EVs isolation from whole tissues can provide a better understanding of intercellular communication in vivo. This underscores the critical need to standardize and optimize protocols for isolating and characterizing EVs from tissues. We have developed a differential centrifugation-based technique to isolate and characterize EVs from whole adipose tissue, which can be potentially applied to other types of tissues. This may help us to better understand the role of EVs in the tissue microenvironment in both diseased and normal conditions. Key features • Isolation of tissue-derived extracellular vesicles from ex vivo culture of visceral adipose tissue or any whole tissue. • Microscopic visualization of extracellular vesicles' morphology without dehydration steps, with minimum effect on their shape. • Flow cytometry approach to characterize the extracellular vesicles using specific protein markers, as an alternative to the time-consuming western blot.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11166539/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141319329","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Gene editing technologies have revolutionized plant molecular biology, providing powerful tools for precise gene manipulation for understanding function and enhancing or modifying agronomically relevant traits. Among these technologies, the CRISPR-Cas9 system has emerged as a versatile and widely accepted strategy for targeted gene manipulation. This protocol provides detailed, step-by-step instructions for implementing CRISPR-Cas9 genome editing in tomato plants, with a specific focus in generating knockout lines for a target gene. For that, the guide RNA should preferentially be designed within the first exon downstream and closer to the start codon. The edited plants obtained are free of transgene cassette for expression of the CRISPR-Cas9 machinery. Key features • Two sgRNAs employed. • Takes 6-12 months to have an edited transgene-free plant. • Setup in tomato.
{"title":"CRISPR-Cas9 Protocol for Efficient Gene Knockout and Transgene-free Plant Generation.","authors":"Enzo A Perk, Ana M Laxalt, Ignacio Cerrudo","doi":"10.21769/BioProtoc.5012","DOIUrl":"10.21769/BioProtoc.5012","url":null,"abstract":"<p><p>Gene editing technologies have revolutionized plant molecular biology, providing powerful tools for precise gene manipulation for understanding function and enhancing or modifying agronomically relevant traits. Among these technologies, the CRISPR-Cas9 system has emerged as a versatile and widely accepted strategy for targeted gene manipulation. This protocol provides detailed, step-by-step instructions for implementing CRISPR-Cas9 genome editing in tomato plants, with a specific focus in generating knockout lines for a target gene. For that, the guide RNA should preferentially be designed within the first exon downstream and closer to the start codon. The edited plants obtained are free of transgene cassette for expression of the CRISPR-Cas9 machinery. Key features • Two sgRNAs employed. • Takes 6-12 months to have an edited transgene-free plant. • Setup in tomato.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11166535/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141319318","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}