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Past Issue in 2024
Volume: 14, Issue: 21
Biological Sciences
PEPITA: Parallelized High-Throughput Quantification of Ototoxicity and Otoprotection in Zebrafish Larvae
Drug-induced hearing injury (ototoxicity) is a common, debilitating side effect of many antibiotic regimens that can be worsened by adverse drug interactions. Such adverse drug interactions are often not detected until after drugs are already on the market because of the difficulty of measuring all possible drug combinations. While in vivo mammalian assays to screen for ototoxic damage exist, they are currently time-consuming, costly, and limited in throughput, which limits their utility in assessing drug interaction outcomes. To facilitate more rapid quantification of ototoxicity and assessment of adverse drug interactions that impact ototoxicity, we have developed a high-throughput workflow we call parallelized evaluation of protection and injury for toxicity assessment (PEPITA). PEPITA uses zebrafish larvae to quantify ototoxic damage and protection. Previous work has shown that hair cells (HCs) in the zebrafish lateral line are very similar to human inner ear HCs, meaning zebrafish are a viable model to test drug-induced ototoxicity. In PEPITA, we expose zebrafish larvae to different combinations of drugs, fluorescently label the HCs, and subsequently use microscopy to quantify the brightness of the fluorescently labeled HCs as an assay for ototoxic damage and hair-cell viability. PEPITA is a reproducible, low-cost, technically accessible, and high-throughput assay. These advantages allow many experiments to be conducted in parallel, paving the way for systematic evaluation of drug-induced hearing injury and other multidrug interactions.
Biophysics
Optimizing Transmembrane Protein Assemblies in Nanodiscs for Structural Studies: A Comprehensive Manual
Membrane protein structures offer a more accurate basis for understanding their functional correlates when derived from full-length proteins in their native lipid environment. Producing such samples has been a primary challenge in the field. Here, we present robust, step-by-step biochemical and biophysical protocols for generating monodisperse assemblies of full-length transmembrane proteins within lipidic environments. These protocols are particularly tailored for cases where the size and molecular weight of the proteins align closely with those of the lipid islands (nanodiscs). While designed for single-span bitopic membrane proteins, these protocols can be easily extended to proteins with multiple transmembrane domains. The insights presented have broad implications across diverse fields, including biophysics, structural biology, and cryogenic electron microscopy (cryo-EM) studies.
Cancer Biology
Accurate Measurement of Cell Number–Normalized Differential Gene Expression in Cells Treated With Retinoic Acid
Genome-wide gene expression analysis is a commonly used method to quantitatively examine the transcriptional signature of any tissue or cell state. Standard bulk cell RNA sequencing (RNA-seq) quantifies RNAs in the cells of the tissue type of interest through massive parallel sequencing of cDNA synthesized from the cellular RNA. The subsequent analysis of global RNA expression and normalization of RNA expression levels between two or more samples generally assumes that cells from all samples produce equivalent amounts of RNA per cell. This assumption may be invalid in cells where MYC or MYCN expression levels are markedly different and thus, overall mRNA expression per cell is altered. Here, we describe an approach for RNA-seq analysis of MYCN-amplified neuroblastoma cells during treatment with retinoic acid, which causes dramatic downregulation of MYCN expression and induces growth arrest and differentiation of the cells. Our procedure employs spiked-in RNA standards added in ratio to the number of cells in each sample prior to RNA extraction. In the analysis of differential gene expression, the expression level of each gene is standardized to the spiked-in RNA standard to accurately assess gene expression levels per cell in conditions of high and low MYCN expression. Our protocol thus provides a step-by-step experimental approach for normalizing RNA-seq expression data on a per-cell-number basis, allowing accurate assessment of differential gene expression in cells expressing markedly different levels of MYC or MYCN.
Developmental Biology
An In Vitro Model of Murine Osteoclast-Mediated Bone Resorption
Osteoclasts are terminally differentiated multinucleated giant cells that mediate bone resorption and regulate skeletal homeostasis under physiological and pathological states. Excessive osteoclast activity will give rise to enhanced bone resorption, being responsible for a wide range of metabolic skeletal diseases, ranging from osteoporosis and rheumatoid arthritis to tumor-induced osteolysis. Therefore, the construction of in vitro models of osteoclast-mediated bone resorption is helpful to better understand the functional status of osteoclasts under (patho)physiological conditions. Notably, it is essential to provide an in vivo–relevant bone substrate that induces osteoclasts to generate authentic resorption lacunae and excavate bone. Here, we summarize the experimental design of a reproducible and cost-effective method, which is suitable for evaluating the regulatory mechanisms and influence of molecular agonists and antagonists as well as therapeutics on osteoclast-mediated bone-resorbing activity.
Generation of Zebrafish Maternal Mutants via Oocyte-Specific Knockout System
Maternal mRNAs and proteins are produced during oogenesis by more than 60% of zebrafish genes. They are indispensable for fertilization and early embryogenesis. Generation and analysis of the maternal mutant is the most direct way to characterize the maternal function of the specific gene. However, due to the lethality of zygotic mutants, the maternal function of most genes in zebrafish remains elusive. Several methods have been developed to circumvent this obstacle, including mRNA rescue, germ-line replacement, oocyte microinjection in situ, mosaic mutation, and bacterial artificial chromosome (BAC)-mediated conditional rescue. Here, we provide an alternative approach to generate zebrafish maternal mutants rapidly and efficiently by introducing four tandem sgRNA expression cassettes into Tg(zpc:zcas9) embryos. This method is more technically feasible and cost- and time-effective than other established methods.
Environmental science
The on-Site Monitoring and Specimen-Making of Ectoparasites on Rodents and Other Small Mammals
The ectoparasites of rodents and other small mammals usually involve five categories of arthropods—fleas, sucking lice, gamasid mites, chigger mites, and occasionally, ticks. These ectoparasites are medically important, serving as vectors for diseases such as plague, murine typhus, scrub typhus, forest encephalitis, Lyme disease, and other zoonoses. Field surveys, collection, and specimen preparation of ectoparasites are crucial for studying taxonomy, faunistics, ecology, and epidemiology. They are also essential for vector surveillance. The present protocol summarizes the on-site monitoring and specimen-making of ectoparasites of rodents and other sympatric small mammals. Besides the collection and specimen preparation of small mammal hosts, the protocol describes in detail the collection, fixation, specimen-making, and taxonomic identification of ectoparasites and provides some monitoring indices. The on-site monitoring indices include the host density index and the infestation indices of ectoparasites (prevalence, mean abundance, mean intensity). The methodologies outlined in this protocol provide technical guidance and references for vector monitoring (surveillance) and control.
Neuroscience
Assessment and Quantification of Foam Cells and Lipid Droplet–Accumulating Microglia in Mouse Brain Tissue Using BODIPY Staining
This paper presents a refined, user-friendly protocol for using boron-dipyrromethene (BODIPY) to assess and quantify foam cells and lipid droplet–accumulating microglia (LDAM) in mouse brain tissue. The protocol aims to enhance existing methodologies by offering precise and efficient evaluation of foam cells and LDAM burden in various neuropathological conditions linked to lipid metabolism and neuroinflammation. A notable challenge in analyzing tissue from mouse models of these neurodegenerative disorders is the interference caused by the autofluorescent molecule lipofuscin. Our protocol addresses this issue with specific steps that effectively distinguish BODIPY fluorescence from lipofuscin autofluorescence, using advanced imaging techniques and filter settings to ensure accurate and reliable analysis. By providing a straightforward and accessible method, this research aims to facilitate the broader adoption of BODIPY-based techniques for detailed foam cell and LDAM analysis in mouse brain tissue, potentially enhancing diagnostic capabilities and deepening our understanding of how these cells contribute to neurodegenerative disease mechanisms.
A Real-Time Approach for Assessing Rodent Engagement in a Nose-Poking Go/No-Go Behavioral Task Using ArUco Markers
Behavioral neuroscience requires precise and unbiased methods for animal behavior assessment to elucidate complex brain–behavior interactions. Traditional manual scoring methods are often labor-intensive and can be prone to error, necessitating advances in automated techniques. Recent innovations in computer vision have led to both marker- and markerless-based tracking systems. In this protocol, we outline the procedures required for utilizing Augmented Reality University of Cordoba (ArUco) markers, a marker-based tracking approach, to automate the assessment and scoring of rodent engagement during an established intracortical microstimulation-based nose-poking go/no-go task. In short, this protocol involves detailed instructions for building a suitable behavioral chamber, installing and configuring all required software packages, constructing and attaching an ArUco marker pattern to a rat, running the behavioral software to track marker positions, and analyzing the engagement data for determining optimal task durations. These methods provide a robust framework for real-time behavioral analysis without the need for extensive training data or high-end computational resources. The main advantages of this protocol include its computational efficiency, ease of implementation, and adaptability to various experimental setups, making it an accessible tool for laboratories with diverse resources. Overall, this approach streamlines the process of behavioral scoring, enhancing both the scalability and reproducibility of behavioral neuroscience research. All resources, including software, 3D models, and example data, are freely available at https://github.com/tomcatsmith19/ArucoDetection.
Semi-Automated Assessment of Long-Term Olfactory Habituation in Drosophila melanogaster Using the Olfactory Arena
Long-lasting memories are a core aspect of an animal’s life. Such memories are characterized by unique molecular mechanisms and often unique circuitry, neither of which are completely understood in vivo. The deep knowledge of the identity and connectivity of neurons of the fruit fly Drosophila melanogaster, as well as the sophisticated genetic tools that allow in vivo perturbations and physiology monitoring, make it a remarkably useful organism in which to investigate the molecular mechanisms of long-term memories. In this protocol, we focus on habituation, a non-associative form of learning, and describe a reliable, semi-automated technique to induce and assess long-term olfactory habituation (LTH) in Drosophila using the olfactory arena, thus providing a method aligned with recent technological progress in behavioral measurement. Prior work has shown that LTH is induced by a 4-day exposure to an odorant and is characterized by a long-lasting (> 24 h) reduction in behavioral response to the exposed odorant, measured using a manual and skill-intensive Y-maze assay. Here, we present a semi-automated protocol for obtaining quantifiable measures of LTH, at the level of detail required for other investigators in the field. Unlike previously described methods, the protocol presented here provides quantitative and detailed behavioral measurements obtained by video recording that can be shared with the scientific community and allows sophisticated forms of offline analysis. We suggest that this procedure has the potential to advance our understanding of molecular and circuit mechanisms of olfactory habituation, its control via neuromodulation, and its interactions with other forms of memory.
Plant Science
Fluorescent Staining and Quantification of Starch Granules in Chloroplasts of Live Plant Cells Using Fluorescein
Plants use CO2, water, and light energy to generate carbohydrates through photosynthesis. During daytime, these carbohydrates are polymerized, leading to the accumulation of starch granules in chloroplasts. The catabolites produced by the degradation of these chloroplast starch granules are used for physiological responses and plant growth. Various staining methods, such as iodine staining, have previously been used to visualize the accumulation of chloroplast starch granules; however, these staining methods cannot be used to image live cells and/or provide confocal images with non-specific signals. In this study, we developed a new imaging method for the fluorescent observation of chloroplast starch granules in living plant cells by staining with fluorescein, a widely available fluorescent dye. This simple staining method, which involves soaking a leaf disk in staining solution, shows high specificity in confocal images. Fluorescent images of the stained tissue allow the cellular starch content of living cells to be quantified with the same level of accuracy as a conventional biochemical method (amyloglucosidase/α-amylase method). Fluorescein staining thus not only enables the easy and clear observation of chloroplast starch granules but also allows for precise quantification in living cells.
Stem Cell
Efficient Gene-Editing in Human Pluripotent Stem Cells Through Simplified Assembly of Adeno-Associated Viral (AAV) Donor Templates
Gene-edited human pluripotent stem cells provide attractive model systems to functionally interrogate the role of specific genetic variants in relevant cell types. However, the need to isolate and screen edited clones often remains a bottleneck, in particular when recombination rates are sub-optimal. Here, we present a protocol for flexible gene editing combining Cas9 ribonucleoprotein with donor templates delivered by adeno-associated virus (AAV) vectors to yield high rates of homologous recombination. To streamline the workflow, we designed a modular system for one-step assembly of targeting vectors based on Golden Gate cloning and developed a rapid protocol for small-scale isolation of AAV virions of serotype DJ. High homology-directed repair (HDR) rates in human pluripotent stem cells (hPSCs), ~70% in ACTB and ~30% in LMNB1, were achieved using this approach, also with short (300 bp) homology arms. The modular design of donor templates is flexible and allows for the generation of conditional and/or complex alleles. This protocol thus provides a flexible and efficient strategy workflow to rapidly generate gene-edited hPSC lines.
Novel Cross-Species Salivary Gland-Parasympathetic Neuron Coculture System
The parasympathetic nervous system is essential for salivary gland development and functionality. Parasympathetic neuron (parasymN) innervation is the main neural network that controls salivary secretion. Therefore, an exclusive model to study parasympathetic neurons and salivary gland tissue circuitry will significantly improve the understanding of the role of parasymN activation on salivary regulation. Harvesting primary rodent parasymNs is challenging due to their body-wide disbursed location. Similarly, the salivary glands are distributed in various locations around and within the oral cavity. Here, we present a coculture model system using human pluripotent stem cell (hPSC)-derived parasymNs and primary mouse von Ebner’s gland cells. We previously reported the first protocol to robustly generate human parasymNs from hPSCs through the Schwann cell precursor (SCP) lineage. The hPSC-parasymNs are functional and have been applied to model several autonomic disorders. We also used a Sox10-Cre::tdTomato (hereafter referred to as RFP) reporter mouse line, which labeled von Ebner’s glands, a type of minor salivary gland connected to the trough of circumvallate and foliate taste papillae. This labeling allowed for visualization and efficient isolation of primary tissues in young adult mice (8–10 weeks). By coculturing the two tissues, human parasymNs control mouse salivary gland cell growth and activation. Both parasymNs and primary salivary gland cells can be frozen and stocked at early stages of differentiation and isolation, making applications easier. This novel coculture model system could also be used to model and study related human diseases in the future, such as dry mouth syndrome.