Biology Methods & Protocols(ISSN: 2396-8923) publishes methodology and protocol papers in the biological sciences. It primarily publishes in the areas of genetics and heredity, biochemistry, molecular biology, ecology, evolutionary biology, marine and freshwater biology and cell biology.
Cold Spring Harbor Protocols is an interdisciplinary journal providing a definitive source of research methods in cell, developmental and molecular biology, genetics, bioinformatics, protein science, computational biology, immunology, neuroscience and imaging. Each monthly issue details multiple essential methods—a mix of cutting-edge and well-established techniques. All protocols are up-to-date and presented in a consistent, easy-to-follow format.
The nervous system of animals can sense and respond to noxious stimuli, which include noxious thermal, chemical, or mechanical stimuli, through a process called nociception. Here, we describe a simple behavioral assay to measure mechanically induced nociceptive responses in Drosophila larvae. This assay tests larval mechanosensitivity to noxious force with calibrated von Frey filaments. First, we explain how to construct and calibrate the customizable von Frey filaments that can be used to deliver reproducible stimuli of a defined force or pressure. Next, we describe how to perform the mechanical nociception assay on third-instar larvae. Through comparison of the responses of genotypes of interest, this assay can be useful for investigation of molecular, cellular, and circuit mechanisms of mechanical nociception. At the molecular level, prior studies have identified the importance of sensory ion channels such as Pickpocket/Balboa, Piezo, dTRPA1, and Painless. At the cellular level, the class IV multidendritic arborizing (md-da) neurons are the main mechanical nociceptor neurons of the peripheral system, but class III and class II md-da have been found to also play a role. At the circuit level, studies have shown that mechanical nociception relies on interneurons of the abdominal ganglia that integrate inputs from these various md-da neuron classes.
In animals, noxious stimuli activate a neural process called nociception. Drosophila larvae perform a rolling escape locomotion behavior in response to nociceptive sensory stimuli. Noxious mechanical, thermal, and chemical stimuli each trigger this same escape response in larvae. The polymodal sensory neurons that initiate the rolling response have been identified based on the expression patterns of genes that are known to be required for nociception responses. The synaptic output of these neurons, known as class IV multidendritic sensory neurons, is required for behavioral responses to thermal, mechanical, and chemical triggers of the rolling escape locomotion. Importantly, optogenetic stimulation of the class IV multidendritic neurons has also shown that the activation of those cells is sufficient to trigger nociceptive rolling. Optogenetics uses light-activated ion channels expressed in neurons of interest to bypass the normal physiological transduction machinery so that the cell may be activated in response to light that is applied by the investigator. This protocol describes an optogenetic technique that uses channelrhodopsin-2 (ChR2) to activate larval nociceptors and trigger nociceptive rolling. First, we explain how to set up the necessary genetic crosses and culture the larval progeny. Next, we describe how to perform the optogenetic nociception assay on third-instar larvae.
Nociception in fruit fly (Drosophila melanogaster) larvae is characterized by a stereotyped escape behavior. When a larva encounters a noxious (potentially harmful) stimulus, it responds by curving its body into a c-shape and rolling in a corkscrew-like manner around its long-body axis. This rolling behavior may serve to quickly remove the larva from the source of the noxious stimulus, and is particularly adaptive to escape from a common natural predator of fruit fly larvae: parasitoid wasps (Leptopilina boulardi). L. boulardi completes its life cycle by using fruit fly larvae as hosts for its offspring. Female wasps sting fly larvae with an ovipositor and lay an egg within the larva. The wasp offspring hatches inside the fly larva, consumes the fly tissues during pupation, and eventually emerges from the pupal case as an adult wasp. Fruit fly larvae respond to oviposition attacks by rolling, which causes the long flexible ovipositor to be wound around the larval body like a spool. This dislodges the wasp and allows the larva to attempt to escape by crawling. Rolling behavior is triggered by the activation of sensory neurons (nociceptors) whose function can inform our understanding of the mechanisms of nociception. In this protocol, we describe a simple behavioral assay to test and measure nociceptive responses in Drosophila larvae during oviposition attacks by female parasitoid wasps. First, we discuss parasitoid wasp husbandry and culturing methods in the laboratory. We then describe how to perform the wasp nociception assay on third-instar fruit fly larvae.
Nociception is the sensory modality by which animals sense stimuli associated with injury or potential tissue damage. When Drosophila larvae encounter a noxious thermal, chemical, or mechanical stimulus, they perform a stereotyped rolling behavior. These noxious stimuli are detected by polymodal nociceptor neurons that tile the larval epidermis. Although several types of sensory neurons feed into the nociceptive behavioral output, the highly branched class IV multidendritic arborization neurons are the most critical. At the molecular level, Drosophila nociception shares many conserved features with vertebrate nociception, making it a useful organism for medically relevant research in this area. Here, we review three larval assays for nociceptive behavior using mechanical stimuli, optogenetic activation, and the naturalistic stimuli of parasitoid wasp attacks. Together, the assays described have been successfully used by many laboratories in studies of the molecular, cellular, and circuit mechanisms of nociception. In addition, the simple nature of the assays we describe can be useful in teaching laboratories for undergraduate students.
Complex behaviors are mediated by a diverse class of neurons and glia produced during development. Both neural stem cell–intrinsic and –extrinsic temporal cues regulate the appropriate number, molecular identity, and circuit assembly of neurons. The Drosophila central complex (CX) is a higher-order brain structure regulating various behaviors, including sensory–motor integration, celestial navigation, and sleep. Most neurons and glia in the adult CX are formed during larval development by 16 Type II neural stem cells (NSCs). Unlike Type I NSCs, which directly give rise to the ganglion mother cells (GMCs), Type II NSCs give rise to multiple intermediate neural progenitors (INPs), and each INP in turn generates multiple GMCs, hence fostering the generation of longer and more diverse lineages. This makes Type II NSCs a suitable model to unravel the molecular mechanisms regulating neural diversity in more complex lineages. In this review, we elaborate on the classification and identification of NSCs based on the types of division adopted and the molecular markers expressed in each type. In the end, we discuss genetic methods for lineage analysis and birthdating. We also explain the temporal expression of stem cell factors and genetic techniques to study how stem cell factors may regulate neural fate specification.
The cuticle is a lipid barrier that covers the air-exposed surfaces of plants. It consists of waxes and cutin, a cell wall–attached lipid polyester of oxygenated fatty acids and glycerol. Unlike waxes, cutin is insoluble in organic solvents, and its composition is typically studied by chemical depolymerization followed by monomer analysis by gas chromatography (GC). Here, we describe a method for the chemical depolymerization of cutin in maize leaves and subsequent compositional analysis of the constituent lipid monomers. The method has been adapted from protocols for cutin analysis developed for Arabidopsis, by both optimizing the amount of leaf tissue used and including a data analysis process specific to the monomers present in maize cutin. The approach uses base-catalyzed transmethylation, which produces fatty acid methyl esters, and silylation, which gives trimethylsilyl ether derivatives of hydroxyl groups for gas chromatographic analysis. For monomer identification, a few representative samples are first analyzed by GC–mass spectrometry (GC-MS). This is then followed by analysis of all replicates by gas chromatography coupled to a flame ionization detector (GC-FID) for monomer quantification, because the flame ionization detector provides a linear response over a wide mass range, is relatively simple to operate, and is more cost-effective to maintain compared to mass spectrometry detectors. Although the protocol bypasses time-consuming cuticle isolation steps by using whole-leaf samples, this means that a fraction of the compounds in the chromatographic profiles do not derive from cutin. Accordingly, we discuss some considerations for the interpretation of the resulting depolymerization products. Our protocol offers specific guidance on preparing maize leaf samples, ensuring reproducible results, and enabling the detection of subtle variations in cutin monomer composition among plant genotypes or developmental stages.
From insects to humans, the nervous system generates complex behaviors mediated by distinct neural circuits that are composed of diverse cell types. During development, the spatiotemporal gene expression of the neural progenitors expands the diversity of neuronal and glial subtypes. Various neural stem cell–intrinsic and –extrinsic gene programs have been identified that are thought to play a major role in generating diverse neuronal and glial cell types. Drosophila has served as an excellent model system for discovering the fundamental principles of nervous system development and function. The sophisticated genetic tools allow us to link the origin and birth timing (the time when a particular neuron is born during development) of neuron types to unique neural stem cells (NSCs) and to a developmental time. In Drosophila, a special class of NSCs called Type II NSCs has adopted a more advanced division mode to generate lineages for the higher-order brain center, the central complex, which is an evolutionarily conserved brain region found in all insects. Type II NSCs, similar to the human outer radial glia, generate intermediate neural progenitors (INPs), which divide many times to produce about eight to 10 progeny. Both Type II NSCs and INPs express distinct transcription factors and RNA-binding proteins that have been proposed to regulate the specification of cell types populating the adult central complex. Here, we describe the recently invented lineage filtering system, called cell class–lineage intersection (CLIn), which enables the tracking and birthdating of the Type II NSC lineages. Using CLIn, one can easily generate clones of different Type II NSCs and identify not only the origins of neurons of interest but also their birth time.
Current Protocols in Chemical Biology (2160-4762) provides clear step-by-step instructions for validated protocols as well as enlightening overviews for researchers who need specialized chemical techniques and tools for their studies of biology and drug design
Cover: Reversible addition-fragmentation chain-transfer (RAFT) polymerization is a controlled radical polymerization process that generates synthetic polymers with controllable molecular weight with low dispersity These RAFT polymers can be conveniently tuned with a versatility that matches the broad needs of materials science including carriers for therapeutic and artificial extracellular matrices for tissue engineering. See Jiang et al. (http://doi.org/10.1002/cpch.85).
The emergence of covalent inhibitors and chemoproteomic probes in translational chemical biology research requires the development of robust biophysical and analytical methods to characterize their complex interactions with target biomolecules. Importantly, these methods must efficiently assess target selectivity and accurately discern noncovalent binding from the formation of resultant covalent adducts. One recently reported covalent chemical tool used in tumor immune oncology, covalent immune recruiters (CIRs), increases the proximity of immune cells and cancer cells, promoting immune recognition and response. Herein we describe biolayer interferometry (BLI) biosensor, flow cytometry, and solution fluorescence-based assay approaches to characterize CIR:antibody binding and CIR-antibody covalent-labeling kinetics. BLI technology, akin to surface plasmon resonance, provides the unique opportunity to investigate molecular binding and labeling kinetics both on a solid surface (Basic Protocol 1) and in solution (Alternate Protocol 1). Here, recruitment of mass-containing proteins to the BLI probe via CIR is measured with high sensitivity and is used as a readout of CIR labeling activity. Further, CIR technology is used to label antibodies with a fluorescent handle. In this system, labeling is monitored via SDS-PAGE with a fluorescence gel imager, where increased fluorescence intensity of a sample reflects increased labeling (Basic Protocol 2). Analysis of CIR:antibody target-specific immune activation is demonstrated with a flow cytometry‒based antibody-dependent cellular phagocytosis (ADCP) assay (Basic Protocol 3). This ADCP protocol may be further used to discern CIR:antibody binding from covalent adduct formation (Alternate Protocol 3). For the protocols described, each method may be used to analyze characteristics of any covalent-tagging or antibody-recruiting small molecule or protein-based technology. © 2020 Wiley Periodicals LLC.
Basic Protocol 1: Determining “on-probe” reaction kinetics of CIR1/CIR4 via biolayer interferometry with Octet RED96
Alternate Protocol 1: Determining “in-solution” reaction kinetics of prostate-specific membrane antigen targeting CIR (CIR3) via biolayer interferometry with Octet RED96
Basic Protocol 2: Reaction kinetics of covalently labeled antibodies via fluorescence SDS-PAGE
Basic Protocol 3: Small molecule‒directed antibody-dependent cellular phagocytosis on live human cells measured via flow cytometry
Alternate Protocol 2: Kinetic analysis of CIR3:antibody labeling via antibody-dependent cellular phagocytosis on flow cytometry
Support Protocol 1: Activation of U937 monocytes with interferon γ
Support Protocol 2: Labeling streptavidin beads with biotinylated prostate-specific membrane antigen receptor
Drug-induced liver injury is an important cause of non-approval in drug development and the withdrawal of already approved drugs from the market. Screening human hepatic cell lines for toxicity has been used extensively to predict drug-induced liver injury in preclinical drug development. Assessing hepatic-cell health with more diverse markers will increase the value of in vitro assays and help predict the mechanism of toxicity. We describe three live cell-based assays using HepG2 cells to measure cell health parameters indicative of hepatotoxicity. The first assay measures cellular ATP levels using luciferase. The second and third assays are multiparametric high-content screens covering a panel of cell health markers including cell count, mitochondrial membrane potential and structure, nuclear morphology, vacuolar density, and reactive oxygen species and glutathione levels. © 2020 Wiley Periodicals LLC.
Basic Protocol 1: Measurement of cellular ATP content
Basic Protocol 2: High-content analysis assay to assess cell count, mitochondrial membrane potential and structure, and reactive oxygen species
Basic Protocol 3: High-content analysis assay to assess nuclear morphology, vacuoles, and glutathione content
Support Protocol 1: Subculturing and maintaining HepG2 cells
Support Protocol 2: Plating HepG2 cell line
Support Protocol 3: Transferring compounds by pin tool
Support Protocol 4: Generating dose-response curves
Cytidine acetyltransferases are an emerging class of nucleic-acid-modifying enzymes responsible for the establishment of N 4-acetylcytidine (ac4C) in RNA. In contrast to histone acetyltransferases, whose activity is commonly studied by western blotting, relatively few methods exist for quickly assessing the activity of cytidine acetyltransferases from a biological sample of interest or the distribution of ac4C across different RNA species. In this protocol, we describe a method for analysis of cellular cytidine acetyltransferase activity using dot- and immuno-northern-blotting-based detection. © 2020 Wiley Periodicals LLC.
Basic Protocol 1: Detection of N 4-Acetylcytidine in RNA by dot blotting
Basic Protocol 2: Visualizing N 4-Acetylcytidine Distribution in RNA by northern blotting
Small molecule microarray (SMM) technology has become a powerful tool used in high-throughput screening for target-based drug discovery. One area in which SMMs have found use is the identification of small molecule ligands for RNA. RNAs with unique secondary or tertiary three-dimensional structures are considered to be attractive targets for small molecules. Complex RNA structures can form hydrophobic pockets suitable for small molecule binding, representing an opportunity for developing novel therapeutics. Our lab has previously taken a target-based approach, screening a single target against many small molecules on an SMM platform. Here, we report a screening protocol for SMMs to investigate multiple RNAs simultaneously using multi-color imaging. By introducing a mixture containing different fluorophore-labeled RNAs, the fluorescence signal of each binding event can be observed simultaneously. Thus, the specificity of a hit compound binding to one RNA target over other highly abundant RNAs (such as tRNA or rRNA) can be easily evaluated. © 2020 Wiley Periodicals LLC.
Basic Protocol: RNA screening on SMMs by multi-color imaging
Support Protocol 1: Preparation of SMM slides
Support Protocol 2: Fluorophore labeling of RNA through maleimide chemistry
Reversible addition-fragmentation chain-transfer (RAFT) polymerization is a commonly used polymerization methodology to generate synthetic polymers. The products of RAFT polymerization, i.e., RAFT polymers, have been widely employed in several biologically relevant areas, including drug delivery, biomedical imaging, and tissue engineering. In this article, we summarize a synthetic methodology to display an azide group at the chain end of a RAFT polymer, thus presenting a reactive site on the polymer terminus. This platform enables a click reaction between azide-terminated polymers and alkyne-containing molecules, providing a broadly applicable scaffold for chemical and bioconjugation reactions on RAFT polymers. We also highlight applications of these azide-terminated RAFT polymers in fluorophore labeling and for promoting organelle targeting capability. © 2020 Wiley Periodicals LLC.
Basic Protocol 1: Synthesis of the azide derivatives of chain transfer agent and radical initiator
Basic Protocol 2: Installation of an azide group on the α-end of RAFT polymers
Alternate Protocol: Installation of an azide group on the ω-end of RAFT polymers
Basic Protocol 3: Click reaction between azide-terminated RAFT polymers and alkyne derivatives
Reverse-polarity activity-based protein profiling (RP-ABPP) is a chemical proteomics approach that uses nucleophilic probes amenable to “click” chemistry deployed into living cells in culture to capture, immunoprecipitate, and identify protein-bound electrophiles. RP-ABPP is used to characterize the structure and function of reactive electrophilic post-translational modifications (PTMs) and the proteins harboring them, which may uncover unknown or novel functions. RP-ABPP has demonstrated utility as a versatile method to monitor the metabolic regulation of electrophilic cofactors, using a pyruvoyl cofactor in S-adenosyl-L-methionine decarboxylase (AMD1), and to discover novel types of electrophilic modifications on proteins in human cells, such as the glyoxylyl modification on secernin-3 (SCRN3). These cofactors cannot be predicted by sequence, and therefore this area is relatively undeveloped. RP-ABPP is the only global, unbiased approach to discover such electrophiles. Here, we describe the utility of these experiments and provide a detailed protocol for de novo discovery, quantitation, and global profiling of electrophilic functionality of proteins. © 2020 The Authors.
Basic Protocol 1: Identification and quantification of probe-reactive proteins
Basic Protocol 2: Characterization of the site of probe labeling
Basic Protocol 3: Determination and quantitation of electrophile structure
Nature Methods (ISSN: 1548-7091) is a monthly journal publishing novel methods and significant improvements to basic life sciences research techniques. All editorial decisions are made by a team of full-time professional editors.
Nature Methods, Published online: 05 May 2025; doi:10.1038/s41592-025-02680-9
This work introduces GLORI 2.0 and 3.0, enabling sensitive m6A quantification from RNA inputs as low as a few hundred cells.Nature Methods, Published online: 01 May 2025; doi:10.1038/s41592-025-02671-w
ATLAS is a rationally designed protein that enables neural circuits to be monosynaptically traced in the anterograde direction from genetically determined starter neurons.Nature Methods, Published online: 01 May 2025; doi:10.1038/s41592-025-02682-7
Scientists who study biodiversity are in the rapid adoption phase of AI. They are finding that what AI can — and can’t — do is shifting rapidly.Nature Methods, Published online: 01 May 2025; doi:10.1038/s41592-025-02677-4
A suite of nontoxic and permeable CarboTag probes in various colors enables live quantitative imaging of a range of plant cell wall characteristics and dynamic, high-resolution mapping of cell wall changes in response to growth or perturbations.Nature Methods, Published online: 01 May 2025; doi:10.1038/s41592-025-02670-x
ATLAS is a tool for circuit tracing, demonstrated here in rodents. It allows anterograde transsynaptic tracing, starting from genetically defined neurons.Nature Methods, Published online: 01 May 2025; doi:10.1038/s41592-025-02659-6
This Review describes sample preparation strategies for time-resolved methods in structural biology, with a focus on cryo-EM and X-ray crystallography, including opportunities for cross-fertilization of ideas between these fields.Nature Methods, Published online: 29 April 2025; doi:10.1038/s41592-025-02699-y
BiaPy: accessible deep learning on bioimagesNature Methods, Published online: 29 April 2025; doi:10.1038/s41592-025-02658-7
This work introduces ChAIR, a droplet-based tri-omic tool that enables the simultaneous profiling of the transcriptome, chromatin accessibility and chromatin conformation in single cells.
Nature Protocols (ISSN: 1750-2799 ) is an online journal of high-quality peer-reviewed protocols for researchers. Protocols are commissioned by the editors or submitted by authors as Presubmission Enquiries. They are presented in a 'recipe' style, providing step-by-step descriptions of procedures that users can take to the lab and immediately apply in their own research. All protocols have been proven to work already, having been used to generate data reported in published research papers.
Nature Protocols, Published online: 29 April 2025; doi:10.1038/s41596-025-01168-2
This is a Protocol extension describing the establishment of human expanded potential stem cell lines from preimplantation embryos or by reprogramming somatic cells and their validation and characterization.Nature Protocols, Published online: 25 April 2025; doi:10.1038/s41596-025-01162-8
Studying the role of protein ubiquitination requires well-characterized ubiquitinated protein derivatives. This protocol describes a semisynthetic approach that can be used to make probes for reader proteins, deubiquitinases and ubiquitin carriers.Nature Protocols, Published online: 25 April 2025; doi:10.1038/s41596-025-01170-8
This protocol describes single-nucleus total RNA-sequencing of formalin-fixed paraffin-embedded samples by using random primers to capture a broad spectrum of RNAs, including nascent and noncoding RNAs, for microfluidics-based droplet barcoding.Nature Protocols, Published online: 24 April 2025; doi:10.1038/s41596-025-01172-6
IgSeqR is a bioinformatic pipeline for de novo assembly and characterization of the tumor immunoglobulin variable and constant region transcripts from RNA sequencing data. Immunoglobulin analysis can provide information on the cell of origin and predict clinical outcomes in B cell cancers.Nature Protocols, Published online: 23 April 2025; doi:10.1038/s41596-025-01160-w
High-throughput computation based on density functional theory (DFT) serves as a cornerstone in materials science. This protocol introduces VASPKIT, a toolkit designed to streamline workflows for the DFT code, Vienna Ab initio Simulation Package.Nature Protocols, Published online: 21 April 2025; doi:10.1038/s41596-025-01161-9
Engineering enzymes capable of performing chemical transformations requires high-throughput assays to screen activity. In this protocol, reactions in cell lysates are analyzed directly by desorption electrospray ionization (DESI) mass spectrometry.Nature Protocols, Published online: 18 April 2025; doi:10.1038/s41596-025-01164-6
Byproducts in the synthesis of [68Ga]Ga-PSMA-11Nature Protocols, Published online: 18 April 2025; doi:10.1038/s41596-025-01163-7
Reply to: Byproducts in the synthesis of [68Ga]Ga-PSMA-11For almost 30 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing.
Methods in Enzymology (ISSN: 0076-6879) a series of scientific publications focused primarily on research methods in biochemistry by Academic Press, created by Sidney P. Colowick and Nathan O. Kaplan, now part of Elsevier. Historically, each volume has centered on a specific topic of biochemistry, such as DNA repair, yeast genetics, or the biology of nitric oxide
Publication date: Available online 22 April 2025
Source: Methods in Enzymology
Author(s): Rosalie Lipsh-Sokolik, Paul J.N. Böhm, Clara Chepkirui, Jörn Piel
Publication date: Available online 17 April 2025
Source: Methods in Enzymology
Author(s): Christina Efthymiou, Sydney Drury, Kenneth Lee
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Source: Methods in Enzymology
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Publication date: Available online 15 April 2025
Source: Methods in Enzymology
Author(s): Vânia Brissos, Paulo Durão, Carolina F. Rodrigues, Eduardo P. Melo, Lígia O. Martins
Publication date: Available online 15 April 2025
Source: Methods in Enzymology
Author(s): José D.D. Cediel-Becerra, Marc G. Chevrette
Publication date: Available online 11 April 2025
Source: Methods in Enzymology
Author(s): Ana Serrano, Juan Carro
Publication date: Available online 2 April 2025
Source: Methods in Enzymology
Author(s): Gabriela Coy, Elliot N. Brajkovich, Elizabeth I. Parkinson
Publication date: Available online 21 March 2025
Source: Methods in Enzymology
Author(s): Shima Nakanishi, John L. Cleveland
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Source: Methods in Enzymology
Author(s): Awatif Alruwaili, Timothy D.H. Bugg
Publication date: Available online 18 March 2025
Source: Methods in Enzymology
Author(s): Lucas F. Ribeiro, Gilvan P. Furtado, Marcos R. Lourenzoni, Richard J. Ward
Journal of visualized experiments [electronic resource] : JoVE
각종 실험 방법을 비디오로 제작하여 제공하는 동영상저널 Biology, Medicine, Immunology and Infection, BioEngineering, Neuroscience 5종 구입