Current protocols最新文献

Precise understanding of temporally controlled protein-protein interactions, localization, and expression is often difficult to achieve using traditional overexpression techniques. Recent advances have made CRISPR-based knock-in approaches efficient, which enables rapid derivation of cells with tagged endogenous proteins. However, the high degree of variability in knock-in efficiency across cell types and gene loci poses challenges, in particular with B lymphocytes, which are refractory to lipid transfection. Here, we present detailed protocols for efficient B lymphoma cell CRISPR/Cas9-mediated knock-in. We address knock-in efficiency in two ways. First, we provide a detailed approach for assessing cutting efficiency to select the most efficient single guide RNA for the gene region of interest. Second, we provide detailed approaches for tagging endogenous proteins with a fluorescent marker or instead for co-expressing them with an unlinked fluorescent marker. Either approach facilitates downstream selection of single-cell or bulk populations with the desired knock-in, particularly when knock-in efficiency is low. The utility of this approach is demonstrated via examples of engineering tags onto endogenous protein N- or C-termini, together with downstream analyses. We anticipate that this workflow can be applied more broadly to other cell types for efficient knock-in into endogenous loci. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Choosing an optimal knock-in target site and single guide RNA (sgRNA) design

Basic Protocol 2: Assessment of Cas9 editing efficiency at the desired B cell genomic knock-in site

Basic Protocol 3: Cloning the sgRNA dual guide construct

Basic Protocol 4: Repair template design and cloning

Basic Protocol 5: Electroporation and selection of engineered B cells

Basic Protocol 6: Single-cell cloning of engineered B cells

Non-symbolic stimuli representing numerosities are invariably associated with continuous magnitudes, complicating the interpretation of experimental studies on numerosity perception. Although various algorithms for experimental numerosity generation have been proposed, they do not consider the quantifiable distribution of values of continuous magnitudes and the degree of numerosity-magnitudes association. Consequently, they cannot thoroughly exclude the possibility of magnitudes integration or strategy switch between different magnitudes in numerical stimulus perception. Here, we introduce a protocol for numerosity generation, animal training, and behavior outcomes analysis that takes the aforementioned issues into consideration. This protocol has been applied to rodents and is applicable to other animals in numerosity studies. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Algorithm for generating non-symbolic numerical stimuli

Alternate Protocol: General algorithm for generating non-symbolic numerical stimuli

Basic Protocol 2: Numerical training and testing for rodents

Extracellular vesicles (EVs) play an important role in cell-cell communication, carrying bioactive molecules including DNA. EV-associated DNA (EV-DNA) has created enormous interest in the field of biomarkers, particularly related to liquid biopsy. However, its analysis is challenging due to the nanoscale structure of EVs, the low abundance of EV-DNA, and surrounding biogenetic debate. Therefore, novel protocols to enhance the accurate detection of EV-DNA are essential to study its role in normal physiology and disease states. Here, we provide two protocols for confirming the presence of EV-DNA from biological samples. In the first protocol, ultrathin sectioning of EVs is combined with immunogold labeling to detect the presence of double-stranded (ds) DNA within the EV lumen using transmission electron microscopy (TEM). In the second protocol, whole-mount EV immunogold labeling allows detailed morphological analysis of EVs and their surface-associated DNA. Using TEM imaging, we have demonstrated that cancer-cell-derived individual EVs exhibit simultaneous positivity for dsDNA and the EV surface protein tetraspanin 9. We believe that this method can be used to label any proteins of interest inside as well as on the surface of EVs. This can aid in the characterization of single EVs and in the identification and verification of EV-associated biomarkers. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: EV isolation from cell-culture-conditioned medium, EV embedding, ultrathin sectioning, labeling, and imaging

Basic Protocol 2: Whole-mount immunolabeling of EV-DNA

NeuroClick is a software tool designed for the in silico execution of azide-alkyne cycloaddition reactions, commonly known as click reactions. We developed this graphical user interface application to expedite the drug discovery process by generating libraries of 1,2,3-triazole compounds. NeuroClick enables users to input reagent SMILES strings, rapidly generating and screening extensive combinatorial libraries at a pace of 10,000 molecules per minute. The software applies stringent criteria to ensure the relevance and accuracy of the generated compounds, excluding molecules without azide groups or those with multiple reactive functional groups to maintain dataset integrity. NeuroClick incorporates advanced filtering options based on Lipinski's rule of five and blood–brain barrier (BBB) permeability predictors, allowing researchers to identify drug-like molecules with potential central nervous system activity. The software's high-throughput and user-friendly interface significantly enhance the efficiency of early-stage drug development by facilitating the exploration of vast chemical spaces and identifying promising lead compounds for further development. This article provides comprehensive guidance on the installation, usage, and features of NeuroClick, ensuring that users can leverage its full potential in their research endeavors. © 2024 Wiley Periodicals LLC.

Basic Protocol: NeuroClick workflow for generating BBB-permeating drugs

Extracellular vesicles (EVs) are small membranous vesicles secreted by cells into their surrounding extracellular environment. Similar to mammalian EVs, plant EVs have emerged as essential mediators of intercellular communication in plants that facilitate the transfer of biological material between cells. They also play essential roles in diverse physiological processes including stress responses, developmental regulation, and defense mechanisms against pathogens. In addition, plant EVs have demonstrated promising health benefits as well as potential therapeutic effects in mammalian health. Despite the plethora of potential applications using plant EVs, their isolation and characterization remains challenging. In contrast to mammalian EVs, which benefit from more standardized isolation protocols, methods for isolating plant EVs can vary depending on the starting material used, resulting in diverse levels of purity and composition. Additionally, the field suffers from the lack of plant EV markers. Nevertheless, three main EV subclasses have been described from leaf apoplasts: tetraspanin 8 positive (TET8), penetration-1-positive (PEN1), and EXPO vesicles derived from exocyst-positive organelles (EXPO). Here, we present an optimized protocol for the isolation and enrichment of small EVs (sEVs; <200 nm) from the apoplastic fluid from Nicotiana benthamiana leaves by ultracentrifugation. We analyze the preparation through transmitted electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting. We believe this method will establish a basic protocol for the isolation of EVs from N. benthamiana leaves, and we discuss technical considerations to be evaluated by each researcher working towards improving their plant sEV preparations. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Isolation and enrichment of small extracellular vesicles (sEVs) from the apoplastic fluid of Nicotiana benthamiana leaves

Short tandem repeats (STRs) and variable-number tandem repeats (VNTRs) are repetitive genomic sequences seen widely throughout the genome. These repeat expansions are currently known to cause ∼60 diseases, with expansions in new loci linked to rare diseases continuing to be discovered. Genome sequencing is an important tool for detecting disease-causing variants and several computational tools have been developed to analyze tandem repeats from genomic data, enabling the genotyping and the identification of expanded alleles. However, guidelines for conducting the analysis of these repeats and, more importantly, for assessing the findings are lacking. Understanding the tools and their technical limitations is important for accurately interpreting the results. This article provides detailed, step-by-step instructions for three key use cases in STR analysis from short-read genome sequencing data, which are also applicable to smaller VNTRs. First, it demonstrates an approach for genotyping known pathogenic loci and the identification of clinically significant expansions. Second, we offer guidance on defining tandem repeat loci and conducting genome-wide genotyping studies, which is also applicable to diploid organisms other than humans. Third, instructions are provided on how to find novel expansions at loci not previously known to be associated with disease, aiding in the discovery of new pathogenic loci. Moreover, we introduce the use of newly-developed helper tools that enable a complete and streamlined tandem repeat analysis protocol by addressing the gaps in current methods. All three protocols are compatible with human hg19, hg38, and the latest telomere-to-telomere (hs1) reference genomes. Additionally, this protocol provides an overview and discussion on how to interpret genotyping results. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Genotyping known pathogenic tandem repeat loci

Alternate Protocol: Genotyping known pathogenic tandem repeat loci with STRipy

Support Protocol 1: Installation of tools and ExpansionHunter catalog modification

Basic Protocol 2: Performing genome-wide genotyping of tandem repeats

Basic Protocol 3: Discovering de novo tandem repeat expansions

Support Protocol 2: Compiling ExpansionHunter Denovo from source code and generating STR profiles

While various methods exist for examining and visualizing the structures of RNA molecules, dimethyl sulfate-mutational profiling and sequencing (DMS-MaPseq) stands out for its simplicity and versatility. This technique has been proven effective for studying RNA structures both in vitro and in complex biological settings. We present an updated protocol of DMS-MaPseq, as well as methodology that enables it to be used for detection of antisense oligonucleotides (ASOs) binding to RNA. By applying this protocol, we successfully characterized the structural ensemble of the HIV1 Rev Response Element (RRE), along with its two alternative structures. The findings align with previously published research validating the accuracy of the method. We also demonstrate the utility of the DMS-MaPseq protocol by resolving and confirming ASO binding at the complementary sites of the P21-associated noncoding RNA DNA damage-activated (PANDA) long non-coding RNA via decreased DMS reactivity. © 2024 Wiley Periodicals LLC. Basic Protocol 1: DMS-MaPseq on HIV1-RRE Basic Protocol 2: DMS-MaPseq on PANDA with ASO probing.

Since their discovery, 3D cell cultures have emerged as powerful tools across various basic, translational research, and industrial discovery projects. One such application is in the physiological and pathophysiological modeling of pancreatic exocrine functions, which addresses critical clinical challenges, including acute and chronic pancreatitis. While several methods now exist for generating epithelial organoids (derived from induced pluripotent, embryonic, or adult stem cells), the advent of patient-derived organoids (PDOs) with controlled polarity has introduced a new frontier in pancreatic research. This advancement has significantly expanded the methodological arsenal available for studying human pancreatic epithelial secretion. In this article, we present basic protocols and a troubleshooting guide for an advanced culture method that results in an apical-to-basal polarity switch. Alongside the protocols, we emphasize a comprehensive cost breakdown, an aspect often challenging to estimate when implementing new techniques. By sharing the technical nuances and financial implications of these protocols, we aim to encourage researchers to transition from rodent models to primary human epithelial cells wherever feasible. This aligns with the U.S. Environmental Protection Agency's efforts to accelerate the translation of significant scientific findings to address major clinical needs. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Establishment and maintenance of pancreatic PDOs Basic Protocol 2: Cryopreservation and thawing of pancreatic PDOs Basic Protocol 3: Inducing polarity switching in pancreatic PDOs.

Cellular immunotherapy has emerged as one of the most potent approaches to treating cancer patients. Adoptive transfer of chimeric antigen receptor (CAR) T cells as well as the use of haploidentical natural killer (NK) cells can induce remission in patients with lymphoma and leukemia. Although the use of CAR T cells has been established, this approach is currently limited for wider use by the risk of severe adverse events, including cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome. Moreover, the risk of triggering graft vs host reactions in settings of allogeneic T cell infusion limits the use to autologous CAR T cells if advanced CRISPR engineering is not applied. In contrast, NK cell-based cancer immunotherapy has emerged as a safe approach even in allogeneic settings. However, efficient transduction of primary blood NK cells with vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped lentivirus commonly used for T cell modification remains challenging. This article presents a detailed method that significantly enhances the transduction efficiency of NK cells by utilizing a short-term culture in cytokine-supplemented medium. It also encompasses the preparation of high-titer and high-quality lentiviral particles for optimal NK cell transduction. Overall, this protocol details the step-by-step culture of NK cells in cytokine-supplemented medium, their transduction with VSV-G lentiviral vectors, and subsequent expansion for functional assays. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Isolation of NK cells from human peripheral blood mononuclear cells (PBMCs)

Basic Protocol 2: NK cell expansion and transduction with lentivirus for generating CAR-NK cells

Support Protocol 1: Plasmid amplification

Support Protocol 2: Lentivirus preparation

Support Protocol 3: Lentivirus titration

In the heart, ion channels generate electrical currents that signal muscle contraction through changes in intracellular calcium concentration, i.e., [Ca²⁺]. The cardiac ryanodine receptor type 2 (RyR2) is the predominant ion channel responsible for increasing intracellular [Ca²⁺] by releasing Ca²⁺ from the sarcoplasmic reticulum (SR). Timely Ca²⁺ release is necessary for appropriate cardiac function, and dysfunction can cause or contribute to life-threatening diseases such as arrhythmia. Quantification of SR-Ca²⁺ release in the form of sparks and waves can provide valuable insight into RyR2 opening, and factors that influence or regulate channel function. Here, we provide a series of protocols that outline processes for (1) obtaining high-quality isolated cardiomyocytes, (2) preparing samples for experimentally investigating factors that influence RyR2 function, and (3) data acquisition and analysis. Notably, our protocols leverage the potency of the recently developed myosin ATPase inhibitor, Mavacamten. This affords the opportunity to characterize the effects of small molecules or reconstituted proteins/enzymes on RyR2-Ca²⁺ release events across a range of [Ca²⁺]. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Cardiomyocyte isolation from mouse

Basic Protocol 2: Preparation of cardiomyocytes for Ca²⁺ imaging

Basic Protocol 3: Confocal microscopy and quantitative Ca²⁺ analysis using SparkMaster 2