Current protocols最新文献

One of the major concerns following cancer treatment is cardiotoxicity. Therefore, it is important to predict potential cardiotoxicity of cancer chemotherapeutics at the preclinical phase. Current models of cardiotoxicity testing involve either cell culture models or rodent models. We developed a simple invertebrate animal model for rapid screening of cardiotoxicity of cancer chemotherapeutics. Daphnia magna (water flea, a crustacean) has a transparent body and a large myogenic heart that can be easily monitored under a microscope. Using this model, we have previously described comparative cardiotoxicity of several kinase inhibitors that were approved for the treatment of multiple cancers. In this article, we describe the step-wise protocols for evaluating the heart rate and survival of D. magna with relevant information on troubleshooting. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Culturing and maintenance of D. magna

Basic Protocol 2: Experimental design for evaluating heart rate of Daphnia

Basic Protocol 3: Long-term effect on Daphnia survival upon drug exposure

Current Protocols is issuing a correction for the following protocol article:

Sozzi, E., Nilsson, F., Kajtez, J., Parmar, M., & Fiorenzano, A. (2022). Generation of human ventral midbrain organoids derived from pluripotent stem cells. Current Protocols, 2, e555. doi: 10.1002/cpz1.555

In the above-referenced article:

The concentration of compound SB431542 in step 14 of Basic Protocol 2 has been corrected. The SB431542 concentration has been changed from 10 nM to 10 µM.

The current version online now includes this correction and may be considered the authoritative version of record.

Genetically modifying mice traditionally involved complex methods of designing and validating targeting constructs, embryonic stem cell electroporation and selection, blastocyst injection, and breeding chimeras for germline transmission. Such arduous steps were best carried out by specialized gene targeting cores in academia or through expensive commercial vendors. Further, the time from initiation to completion of a project often took at least 1 year and, in some cases, much longer (or never), with no guarantees of success. The RNA-programmable CRISPR system of gene editing has greatly streamlined the generation of gene modifications (e.g., small substitutions, insertions, and deletions) in the mouse with high rates of success. Several editing platforms exist for gene/genome targeting in mice and other animal models previously difficult or impossible to alter. Here, we provide a simplified method of generating genetically modified mice using the prime editing platform. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Design, cloning, and synthesis of engineered pegRNA (epegRNA)

Basic Protocol 2: Microinjection of PE2 components into mouse zygote

Basic Protocol 3: Genotyping founder mice and breeding for germline transmission

Three-dimensional (3D) printing promises a revolution in laboratory creativity by enabling rapid prototyping, broader availability of scientific apparatuses, and transformative scientific workflows. We believe all chemistry and biology laboratories should equip themselves with one or more 3D printers and a critical mass of scientists trained to operate them. This overview surveys the techniques, intricacies, and pitfalls associated with 3D printing of functional parts, including measurements, computer-aided design, slicing, limitations of 3D printing, troubleshooting, tips for tricky filaments, and 3D printer maintenance. A flow cells are essential tools in chemistry and biology laboratories, we discuss techniques relevant to the construction of watertight 3D-printed parts. Finally, we articulate a set of principles required for reporting 3D-printed innovations to improve the field's reproducibility and encourage iterative improvements by other scientists. Ideally, authors, peer reviewers, and editors will adopt these principles. We hope these protocols inspire a new generation of publications applying 3D printing in chemistry and biology—especially highly reproducible inventions with the requisite detail and associated documentation. Such reports will facilitate broad adoption and creative iteration of the most innovative designs, thus accelerating discovery in chemistry and biology. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Estrogen receptor alpha (ERα) is a nuclear receptor that is expressed mainly in the breast, uterus, and ovary, among several other organs. ERα plays important roles in reproduction, mammary gland formation, and glucose homeostasis. Disruption of ERα may result in adverse outcomes, such as cancer, impaired fertility, and abnormal fetal growth. Therefore, identifying compounds that modulate ERα is of great interest due to their potential-endocrine disrupting capability and pharmaceutical applications. To rapidly test tens of thousands of compounds, high-throughput screening assays are essential. Here, we describe high-throughput screening methods, including plating and treatment of cells in 384-well and 1536-well plates and analysis of the resulting data. The two cell lines used, MCF7-VM7Luc4E2 and HEK293-ERα-bla, have been described previously. MCF7-VM7Luc4E2 cells are a stable luciferase reporter gene cell line expressing full-length endogenous estrogen receptor in the MCF7 cell line background, and HEK293-ERα-bla cells stably express an ERα ligand-binding domain/GAL4 DNA-binding domain fusion regulating a UAS β-lactamase reporter gene. These cell lines can be used to identify and confirm ERα modulators. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Establishment of a high-throughput ERα reporter gene assay with luminescence readout to identify activators and inhibitors of estrogen receptor α

Basic Protocol 2: Use of an orthogonal assay with fluorescence readout to confirm potential estrogen receptor activators or inhibitors

Flow cytometry is an inherently fluidic process that flows particles on a one-by-one basis through a sensing region to discretely measure their optical and physical properties. It can be used to analyze particles ranging in size from nanoparticles to whole organisms (e.g., zebrafish). It has particular value for blood analysis, and thus most instruments are fluidically optimized for particles that are comparable in size to a typical blood cell. The principles of fluid dynamics allow for particles of such size to be precisely positioned in flow as they pass through sensing regions that are tens of microns in length at linear velocities of meters per second. Such fluidic systems enable discrete analysis of cell-sized particles at rates approaching 100 kHz. For larger particles, the principles of fluidics greatly reduce the achievable rates, but such high rates of data acquisition for cell-sized particles allow rapid collection of information on many thousands to millions of cells and provides for research and clinical measurements of both rare and common cell populations with a high degree of statistical confidence. Additionally, flow cytometers can accurately count particles via the use of volumetric sample delivery and can be coupled with high-throughput sampling technologies to greatly increase the rate at which independent samples can be delivered to the system. Due to the combination of high analysis rates, sensitive multiparameter measurements, high-throughput sampling, and accurate counting, flow cytometry analysis is the gold standard for many critical applications in clinical, research, pharmaceutical, and environmental areas. Beyond the power of flow cytometry as an analytical technique, the fluidic pathway can be coupled with a sorting mechanism to collect particles based on desired properties. We present an overview of fluidic systems that enable flow cytometry–based analysis and sorting. We introduce historical approaches, explanations of commonly implemented fluidics, and brief discussions of potential future fluidics where appropriate. © 2024 Wiley Periodicals LLC.

The International Molecular Exchange Consortium (IMEx) has evolved into a vital partnership of open resources dedicated to curating molecular interaction data from the scientific literature. This consortium, which includes IntAct, MINT, MatrixDB, and DIP, is a collaborative effort with a central mission of aggregating detailed molecular interaction experimental evidence in a machine-readable format, supported by controlled vocabularies and standard ontologies. The IntAct molecular interaction database (www.ebi.ac.uk/intact), as an IMEx partner, serves as a valuable portal for accessing IMEx data through user-friendly search options and an array of interactive filters. The resource currently hosts an extensive repository of 1,293,508 binary interactions meticulously captured from 75,098 experiments documented in 23,366 publications (as of the February 2024 release), with this corpora being added to by regular data releases. IMEx curation policy has consistently prioritized a fine-grained data and curation model, with a focus on capturing the relevant experimental details essential for interpreting molecular interaction data effectively. Our curation process is designed to support the generation of interactomes tailored to contexts such as disease-specific or tissue-/cell-type-specific interactomes. These interactions are ranked according to a scoring system based on the Proteomics Standard Initiative Molecular Interaction (PSI MI) standards. This scoring system allows users to assess the degree of confidence in binary interactions, enhancing the value of the data. The resource provides insights into the nature of relationships among interacting partners as defined by the experimental setup and the associated biological context. Interactive filters enable users to navigate these rich, multilayered data, promoting a deeper understanding of biological complexity. Additionally, the IntAct website fosters the creation of networks for collaborative analyses by the scientific community. The recent transformation of the IntAct website, supported by a graph-type database, empowers users to execute custom queries tailored to their specific research interests. This article illustrates the diverse levels of annotations available for interactions and the multiple search options at users’ disposal to access data of interest. © 2024 European Molecular Biology Laboratory, European Bioinformatics Institute. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Using Quick Search, network visualization, and filters

Support Protocol: Accessing fine annotations from intact: Unlocking the molecular details

Alternate Protocol: Using batch search: Querying multiple interactors

Basic Protocol 2: Using advanced search: Precision and customization

Three-dimensional (3D) cerebral cortical organoids are popular in vitro cellular model systems widely used to study human brain development and disease, compared to traditional stem cell–derived methods that use two-dimensional (2D) monolayer cultures. Despite the advancements made in protocol development for cerebral cortical organoid derivation over the past decade, limitations due to biological, mechanistic, and technical variables remain in generating these complex 3D cellular systems. Building from our previously established differentiation system, we have made modifications to our existing 3D cerebral cortical organoid protocol that resolve several of these technical and biological challenges when working with diverse groups of human induced pluripotent stem cell (hiPSC) lines. This improved protocol blends a 2D monolayer culture format for the specification of neural stem cells and expansion of neuroepithelial progenitor cells with a 3D system for improved self-aggregation and subsequent organoid development. Furthermore, this “hybrid” approach is amenable to both an accelerated cerebral cortical organoid protocol as well as an alternative long-term differentiation protocol. In addition to establishing a hybrid technical format, this protocol also offers phenotypic and morphological characterization of stage-specific cellular profiles using antibodies and fluorescent-based dyes for live cell imaging. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: hiPSC-based 2D monolayer specification into neural stem cells (NSCs)

Basic Protocol 2: Serial passaging and 2D monolayer expansion of neuroepithelial progenitor cells (NPCs)

Support Protocol 1: Direct cryopreservation and rapid thawing of NSCs and NPCs

Basic Protocol 3: Bulk aggregation of 3D neurospheres and accelerated cerebral cortical organoid differentiation

Alternate Protocol 1: Bulk aggregation of 3D neurospheres and long-term cerebral cortical organoid differentiation

Support Protocol 2: High-throughput 3D neurosphere formation and 2D neurosphere migration assay

Support Protocol 3: LIVE/DEAD stain cell imaging assay of 3D neurospheres

Support Protocol 4: NeuroFluor NeuO live cell dye for 3D cerebral cortical organoids

High-quality DNA with sufficient yield is the goal of DNA extraction protocols. We present an optimized, cost-effective method for extracting next-generation sequencing (NGS)-quality genomic DNA from Bacillus and Clostridium species using the chloroform-isoamyl approach. The protocol involves two main procedures: cultivation of the bacteria under appropriate conditions, followed by DNA extraction through cell lysis, phase separation, DNA precipitation, and cleanup. This method has been successfully applied to several hundred strains of Bacillus and Clostridium species in our laboratory, including B. cereus, B. licheniformis, C. sporogenes, and C. tyrobutyricum, demonstrating its efficacy and reliability for producing high-quality DNA that meets NGS quality control standards. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Culturing Bacillus and Clostridium species

Basic Protocol 2: DNA extraction © 2024 by John Wiley & Sons, Inc.

The liver's role in the biotransformation of chemicals is critical for both augmented toxicity and detoxification. However, there has been a significant lack of effort to integrate biotransformation into in vitro neurotoxicity testing. Traditional in vitro neurotoxicity testing systems are unable to assess the qualitative and quantitative differences between parent chemicals and their metabolites as they would occur in the human body. As a result, traditional in vitro toxicity screening systems cannot incorporate hepatic biotransformation to predict the neurotoxic potential of chemical metabolites. To bridge this gap, a high-throughput, metabolism-mediated neurotoxicity testing system has been developed, which combines metabolically competent HepaRG cell spheroids with a three-dimensional (3D) culture of ReNcell VM human neural progenitor cell line. The article outlines protocols for generating HepaRG cell spheroids using an ultralow attachment (ULA) 384-well plate and for cultivating ReNcell VM in 3D on a 384-pillar plate with sidewalls and slits (384PillarPlate). Metabolically sensitive test compounds are introduced into the ULA 384-well plate containing HepaRG spheroids and then tested with 3D-cultured ReNcell VM on the 384PillarPlate. This configuration permits the in situ generation of metabolites by HepaRG cells and their subsequent testing on neurospheres. By analyzing cell viability data, researchers can determine the IC₅₀ values for each compound, thus evaluating metabolism-mediated neurotoxicity. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: HepaRG spheroid culture in an ultralow attachment (ULA) 384-well plate and the assessment of drug-metabolizing enzyme (DME) activities

Basic Protocol 2: 3D neural stem cell (NSC) culture on a 384PillarPlate and compound treatment for the assessment of metabolism-mediated neurotoxicity

Basic Protocol 3: Image acquisition, processing, and data analysis