Current protocols最新文献

{"title":"Quantification of Sarcoplasmic Reticulum Ca2+ Release in Primary Ventricular Cardiomyocytes","authors":"Md Nure Alam Afsar,&nbsp;Mahmuda Akter,&nbsp;Christopher Y. Ko,&nbsp;Vasco Sequeira,&nbsp;Yusuf Olgar,&nbsp;Christopher N. Johnson","doi":"10.1002/cpz1.70048","DOIUrl":"https://doi.org/10.1002/cpz1.70048","url":null,"abstract":"<p>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.</p><p><b>Basic Protocol 1</b>: Cardiomyocyte isolation from mouse</p><p><b>Basic Protocol 2</b>: Preparation of cardiomyocytes for Ca²⁺ imaging</p><p><b>Basic Protocol 3</b>: Confocal microscopy and quantitative Ca²⁺ analysis using SparkMaster 2</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 11","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142540983","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"PhyKIT: A Multitool for Phylogenomics","authors":"Jacob L. Steenwyk,&nbsp;Gemma I. Martínez-Redondo,&nbsp;Thomas J. Buida III,&nbsp;Emile Gluck-Thaler,&nbsp;Xing-Xing Shen,&nbsp;Toni Gabaldón,&nbsp;Antonis Rokas,&nbsp;Rosa Fernández","doi":"10.1002/cpz1.70016","DOIUrl":"https://doi.org/10.1002/cpz1.70016","url":null,"abstract":"<p>Multiple sequence alignments and phylogenetic trees are rich in biological information and are fundamental to research in biology. PhyKIT is a tool for processing and analyzing the information content of multiple sequence alignments and phylogenetic trees. Here, we describe how to use PhyKIT for diverse analyses, including (i) constructing a phylogenomic supermatrix, (ii) detecting errors in orthology inference, (iii) quantifying biases in phylogenomic data sets, (iv) identifying radiation events or lack of resolution using gene support frequencies, and (v) conducting evolution-based screens to facilitate gene function prediction. Several PhyKIT functions that streamline multiple sequence alignment and phylogenetic processing—such as renaming FASTA entries or tree tips—are also discussed. These protocols demonstrate how simple command-line operations in the unified framework of PhyKIT facilitate diverse phylogenomic data analysis and processing, from supermatrix construction and diagnosis to gaining clues about gene function. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Installing PhyKIT and syntax for usage</p><p><b>Basic Protocol 2</b>: Constructing a phylogenomic supermatrix</p><p><b>Basic Protocol 3</b>: Detecting anomalies in orthology relationships</p><p><b>Basic Protocol 4</b>: Quantifying biases in phylogenomic data matrices and related measures</p><p><b>Basic Protocol 5</b>: Identifying polytomies</p><p><b>Basic Protocol 6</b>: Assessing gene-gene coevolution as a genetic screen</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 11","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70016","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142540947","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}

{"title":"Transmission Electron Microscopy of Coral Tissue","authors":"Erin Papke,&nbsp;Grace E. Kennedy,&nbsp;Elizabeth Elliott,&nbsp;Alison Taylor,&nbsp;Bradley B. Tolar,&nbsp;Blake Ushijima","doi":"10.1002/cpz1.70033","DOIUrl":"https://doi.org/10.1002/cpz1.70033","url":null,"abstract":"<p>Coral reefs are invaluable ecosystems that are under threat from various anthropogenic stressors. There has been a recent increase in the diagnostic tools utilized to understand how these threats impact coral reef health. Unfortunately, the application of diagnostic tools like transmission electron microscopy (TEM) is not as standardized or developed in coral research as in other research fields. Utilizing TEM in conjunction with other diagnostic methods can aid in understanding the impact of these stressors on the cellular level because TEM offers valuable insight into the structures and microsymbionts associated with coral tissue that cannot be obtained with a conventional light microscope. Additionally, a significant amount of coral tissue ultrastructure has not yet been extensively described, causing a considerable gap in our understanding of cellular structures that could relate to the immune response, cellular function, or symbioses. Moreover, additional standardization is needed for TEM in coral research to increase comparability and reproducibility of findings across studies. Here, we present standardized TEM sample fixation, embedding, and sectioning techniques for coral studies that ensure consistent ultrastructural preservation and minimize artifacts, enhancing the reliability and accuracy of TEM observations. We also demonstrate that these TEM protocols allow for the observation and quantification of bacterial and viral-like particles within the coral tissue as well as the endosymbiotic microalgae, potentially providing insight into their interactions within coral cells and how they relate to overall coral health and resilience. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Primary fixation</p><p><b>Basic Protocol 2</b>: Decalcification</p><p><b>Basic Protocol 3</b>: Sample dissection, secondary fixation, dehydration, and embedding</p><p><b>Basic Protocol 4</b>: Sectioning and grid staining</p><p><b>Basic Protocol 5</b>: Imaging</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 11","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70033","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142540949","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}

{"title":"Generation of Barcode-Tagged Vibrio fischeri Deletion Strains and Barcode Sequencing (BarSeq) for Multiplex Strain Competitions","authors":"Hector L. Burgos,&nbsp;Mark J. Mandel","doi":"10.1002/cpz1.70024","DOIUrl":"10.1002/cpz1.70024","url":null,"abstract":"<p><i>Vibrio fischeri</i> is a model mutualist for studying molecular processes affecting microbial colonization of animal hosts. We present a detailed protocol for a barcode sequencing (BarSeq) approach that combines targeted gene deletion with short-read sequencing technology to enable studies of mixed bacterial populations. This protocol includes wet lab steps to plan and produce the deletions, approaches to scale up mutant generation, protocols to prepare and conduct the strain competition, library preparation for sequencing on an Illumina iSeq 100 instrument, and data analysis with the barseq python package. Aspects of this protocol could be readily adapted for tagging wild-type <i>V. fischeri</i> strains with a neutral barcode for examination of population dynamics or BarSeq analyses in other species. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Production of the <i>erm-bar</i> DNA</p><p><b>Basic Protocol 2</b>: Generation of a targeted and barcoded deletion strain of <i>V. fischeri</i></p><p><b>Alternate Protocol</b>: Parallel generation of multiple barcode-tagged <i>V. fischeri</i> deletion strains</p><p><b>Basic Protocol 3</b>: Setting up mixed populations of barcode-tagged strains</p><p><b>Basic Protocol 4</b>: Performing a competitive growth assay</p><p><b>Basic Protocol 5</b>: Amplicon library preparation and equimolar pooling</p><p><b>Basic Protocol 6</b>: Sequencing on Illumina iSeq 100</p><p><b>Basic Protocol 7</b>: BarSeq data analysis</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 10","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70024","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142515177","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}

{"title":"GEDI: An R Package for Integration of Transcriptomic Data from Multiple Platforms for Bioinformatics Applications","authors":"Mathias N. Stokholm,&nbsp;Maria B. Rabaglino,&nbsp;Haja N. Kadarmideen","doi":"10.1002/cpz1.70046","DOIUrl":"10.1002/cpz1.70046","url":null,"abstract":"<p>Transcriptomic data is often expensive and difficult to generate in large cohorts relative to genomic data; therefore, it is often important to integrate multiple transcriptomic datasets from both microarray- and next generation sequencing (NGS)-based transcriptomic data across similar experiments or clinical trials to improve analytical power and discovery of novel transcripts and genes. However, transcriptomic data integration presents a few challenges including reannotation and batch effect removal. We developed the Gene Expression Data Integration (GEDI) R package to enable transcriptomic data integration by combining existing R packages. With just four functions, the GEDI R package makes constructing a transcriptomic data integration pipeline straightforward. Together, the functions overcome the complications in transcriptomic data integration by automatically reannotating the data and removing the batch effect. The removal of the batch effect is verified with principal component analysis and the data integration is verified using a logistic regression model with forward stepwise feature selection. To demonstrate the functionalities of the GEDI package, we integrated five bovine endometrial transcriptomic datasets from the NCBI Gene Expression Omnibus. These transcriptomic datasets were from multiple high-throughput platforms, namely, array-based Affymetrix and Agilent platforms, and NGS-based Illumina paired-end RNA-seq platform. Furthermore, we compared the GEDI package to existing tools and found that GEDI is the only tool that provides a full transcriptomic data integration pipeline including verification of both batch effect removal and data integration for downstream genomic and bioinformatics applications. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: ReadGE, a function to import gene expression datasets</p><p><b>Basic Protocol 2</b>: GEDI, a function to reannotate and merge gene expression datasets</p><p><b>Basic Protocol 3</b>: BatchCorrection, a function to remove batch effects from gene expression data</p><p><b>Basic Protocol 4</b>: VerifyGEDI, a function to confirm successful integration of gene expression data</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 10","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70046","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142515176","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}

{"title":"Synthesis of N3-Methyluridine- and 2′-O-Alkyl/2′-Fluoro-N3-Methyluridine-Modified Phosphoramidites and Their Incorporation into DNA and RNA Oligonucleotides","authors":"Avijit Sahoo,&nbsp;Kiran R. Gore","doi":"10.1002/cpz1.70039","DOIUrl":"10.1002/cpz1.70039","url":null,"abstract":"<p>In this article, we describe the synthesis of <i>N</i>³-methyluridine (m³U) and 2′-<i>O</i>-alkyl/2′-fluoro-<i>N</i>³-methyluridine (2′-<i>O</i>-alkyl/2′-F-m³U) phosphoramidites as well as their incorporation into a 14-mer DNA and RNA oligonucleotide sequence. Synthesis of the 2′-<i>O</i>-alkyl-m³U phosphoramidite starts with commercially available uridine to achieve a tritylated m³U intermediate, followed by 2′-<i>O</i>-alkylation and finally phosphitylation. Synthesis of the 2′-F-m³U phosphoramidite is obtained from a commercially available 2′-F-uridine nucleoside. These phosphoramidite monomers are compatible with DNA and RNA oligonucleotide synthesis using conventional phosphoramidite chemistry. This strategy offers efficient synthetic access to various modifications at the 2′-position of m³U that can be employed in numerous nucleic acid–based therapeutic applications, including antisense technologies, small interfering RNAs, CRISPR-Cas9, and aptamers. The data presented in this article are based on our previously published reports. © 2024 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Synthesis of 2′-<i>O</i>-alkyl-<i>N</i>³-methyluridine analogs and their corresponding phosphoramidites</p><p><b>Alternate Protocol 1</b>: Synthesis of 2′-<i>O</i>-TBDMS-<i>N</i>³-methyluridine and its phosphoramidite</p><p><b>Alternate Protocol 2</b>: Synthesis of 2′-fluoro-<i>N</i>³-methyluridine and its phosphoramidite</p><p><b>Basic Protocol 2</b>: Solid-phase synthesis of <i>N</i>³-methyluridine-modified DNA and RNA oligonucleotides</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 10","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142515179","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The TELCoMB Protocol for High-Sensitivity Detection of ARG-MGE Colocalizations in Complex Microbial Communities","authors":"Jonathan E. Bravo,&nbsp;Ilya Slizovskiy,&nbsp;Nathalie Bonin,&nbsp;Marco Oliva,&nbsp;Noelle Noyes,&nbsp;Christina Boucher","doi":"10.1002/cpz1.70031","DOIUrl":"10.1002/cpz1.70031","url":null,"abstract":"<p>Understanding the genetic basis of antimicrobial resistance is crucial for developing effective mitigation strategies. One necessary step is to identify the antimicrobial resistance genes (ARGs) within a microbial population, referred to as the resistome, as well as the mobile genetic elements (MGEs) harboring ARGs. Although shotgun metagenomics has been successful in detecting ARGs and MGEs within a microbiome, it is limited by low sensitivity. Enrichment using cRNA biotinylated probes has been applied to address this limitation, enhancing the detection of rare ARGs and MGEs, especially when combined with long-read sequencing. Here, we present the TELCoMB protocol, a Snakemake workflow that elucidates resistome and mobilome composition and diversity and uncovers ARG-MGE colocalizations. The protocol supports both short- and long-read sequencing and does not require enrichment, making it versatile for various genomic data types. TELCoMB generates publication-ready figures and CSV files for comprehensive analysis, improving our understanding of antimicrobial resistance mechanisms and spread. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Installing TELCOMB Locally</p><p><b>Alternate Protocol</b>: Installing TELCOMB on a SLURM Cluster</p><p><b>Basic Protocol 2</b>: Data Preprocessing</p><p><b>Basic Protocol 3</b>: Calculation of Resistome Distribution and Composition</p><p><b>Basic Protocol 4</b>: Identification of ARG-MGE Colocalizations</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 10","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70031","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142515180","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}

{"title":"Recording and Interpretation of Active Calcium Transients in Induced Pluripotent Stem Cell-Derived Cardiomyocytes","authors":"Maedeh Mozneb,&nbsp;Jemima Moses,&nbsp;Madelyn Arzt,&nbsp;Sean Escopete,&nbsp;Arun Sharma","doi":"10.1002/cpz1.70030","DOIUrl":"10.1002/cpz1.70030","url":null,"abstract":"<p>Calcium plays a pivotal role in the excitation-contraction coupling process in cardiomyocytes, a critical multi-parametric event leading to rhythmic contraction. Over the past few decades, calcium signaling in cardiomyocytes has been extensively studied in cardiovascular sciences. However, a standard methodology is needed not only to trace the calcium within cells but also to remove signal processing biases and to accurately interpret the features of calcium transient signals in relation to cardiomyocyte electrophysiology. This article outlines the use of genetically encoded calcium indicator (GCaMP) human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to record calcium transients. These cells express a green fluorescent signal when calcium binds to intracellular calmodulin, a key regulator of calcium signaling. The extraction and processing of calcium transient waveforms are performed using ImageJ and MATLAB software. Key features of these waveforms are then identified and categorized based on their physiological relevance to cardiomyocyte function. Additionally, this work includes a Support Protocol for the successful replating of cardiomyocytes onto non-traditional culture platforms, such as metallic sensors and polymer-based substrates, to facilitate data multiplexing. The three Basic Protocols outlined here provide a comprehensive approach for maintaining, expanding, and differentiating the GCaMP hiPSCs, video recording of calcium transients, and the subsequent signal extraction, preprocessing, analysis, and data visualization. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Maintenance, expansion, and differentiation of genetically encoded calcium indicator hiPSCs</p><p><b>Support Protocol</b>: Replating GCaMP hiPSC-CMs for stimulation and multielectrode array studies</p><p><b>Basic Protocol 2</b>: Video recording from calcium transients of GCaMP hiPSC-CMs</p><p><b>Basic Protocol 3</b>: Signal extraction, preprocessing, analysis, and data visualization</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 10","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70030","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142515178","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}

{"title":"Correction: Exome Sequencing Starting from Single Cells","authors":"Ioanna Andreou,&nbsp;Markus Storbeck,&nbsp;Peter Hahn,&nbsp;Samuel Rulli,&nbsp;Eric Lader","doi":"10.1002/cpz1.70052","DOIUrl":"10.1002/cpz1.70052","url":null,"abstract":"<p><i>Current Protocols</i> is issuing corrections for the following protocol article:</p><p>Andreou, I., Storbeck, M., Hahn, P., Rulli, S., &amp; Lader, E. (2024). Exome sequencing starting from single cells. <i>Current Protocols</i>, <i>4</i>, e70017. doi: 10.1002/cpz1.70017</p><p>In the above-referenced article:</p><p>A new Table 10 has been added and the existing Table 10 has been renumbered as Table 12.</p><p>The existing Tables 12 to 14 have been renumbered as Tables 13 to 15.</p><p>The current version online now includes these corrections and may be considered the authoritative version of record.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 10","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70052","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142515175","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}

{"title":"Kindling Models of Epileptogenesis for Developing Disease-Modifying Drugs for Epilepsy","authors":"Doodipala Samba Reddy,&nbsp;Abhinav Vadassery,&nbsp;Sreevidhya Ramakrishnan,&nbsp;Tanveer Singh,&nbsp;Bryan Clossen,&nbsp;Xin Wu","doi":"10.1002/cpz1.70020","DOIUrl":"10.1002/cpz1.70020","url":null,"abstract":"<p>Kindling models are widely used animal models to study the pathobiology of epilepsy and epileptogenesis. These models exhibit distinctive features whereby sub-threshold stimuli instigate the initial induction of brief focal seizures. Over time, the severity and duration of these seizures progressively increase, leading to a fully epileptic state, which is marked by consistent development of generalized tonic-clonic seizures. Kindling involves focal stimulation via implanted depth electrodes or repeated administration of chemoconvulsants such as pentylenetetrazol. Comparative analysis of preclinical and clinical findings has confirmed a high predictive validity of fully kindled animals for testing novel antiseizure medications. Thus, kindling models remain an essential component of anticonvulsant drug development programs. This article provides a comprehensive guide to working protocols, testing of therapeutic drugs, outcome parameters, troubleshooting, and data analysis for various electrical and chemical kindling epileptogenesis models for new therapeutic development and optimization. The use of pharmacological agents or genetically modified mice in kindling experiments is valuable, offering insights into the impact of a specific target on various aspects of seizures, including thresholds, initiation, spread, termination, and the generation of a hyperexcitable network. These kindling epileptogenesis paradigms are helpful in identifying mechanisms and disease-modifying interventions for epilepsy. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Hippocampal kindling</p><p><b>Basic Protocol 2</b>: Amygdala kindling</p><p><b>Basic Protocol 3</b>: Rapid hippocampal kindling</p><p><b>Basic Protocol 4</b>: Chemical kindling</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 10","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11498896/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142483996","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}