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The stop signal is produced in response to negative experiences at the food source and inhibits honey bee (Apis mellifera) waggle dancing. Here, we present a protocol for measuring the effects of an inhibitory signal associated with danger on honey bee dopamine levels. We describe steps for observing honey bee colonies, training them with artificial nectar, and simulating hornet attacks. We then detail procedures for recording waggle dancing and stop signals and measuring brain dopamine levels during different treatments. For complete details on the use and execution of this protocol, please refer to Dong et al.1.

Electroporation is a technique to introduce DNA constructs into cells using electric current. Here, we present a protocol to electroporate DNA plasmids into Ciona robusta embryos at the 1-cell stage. We describe steps for setting up and conducting electroporation. We then detail procedures for collecting, fixing, and mounting embryos and counting expression. This protocol can be used to study the expression of enhancers via reporter assays, manipulating cells using genes or modified genes such as dominant negatives, and genome editing. For complete details on the use and execution of this protocol, please refer to Song, et al.1.

Whole-organism-based screen holds promise for discovering biologically active compounds. However, high-content imaging is challenging due to the difficulty of positioning live animals and individual variability of neuron counts. Here, we present a protocol to identify neuroprotective compounds for dopaminergic neurons in zebrafish using an image-based small-molecule screen. We describe steps for raising larvae, agarose embedding, and treatment to induce neurodegeneration. We then detail procedures for live confocal imaging, image processing, and data analysis. For complete details on the use and execution of this protocol, please refer to Kim et al. (2021).1.

Transmission electron microscopy has been essential for understanding cell biology for over six decades. Volume electron microscopy tools, such as serial block face and focused ion beam scanning electron microscopy acquisition, brought a new era to ultrastructure analysis. 'Array Tomography' (AT) refers to sequential image acquisition of resin‐embedded sample sections on a large support (coverslip, glass slide, silicon wafers) for immunolabelling with multiple fluorescent labels, occasionally combined with ultrastructure observation. Subsequently, the term was applied to generating and imaging a series of sections to acquire a 3D representation of a structure using scanning electron microscopy (SEM). Although this is a valuable application, the potential of AT is to facilitate many tasks that are difficult or even impossible to obtain by Transmission Electron Microscopy (TEM). Due to the straightforward nature and versatility of AT sample preparation and image acquisition, the technique can be applied practically to any biological sample for selected sections or volume electron microscopy analysis. Furthermore, in addition to the benefits described here, AT is compatible with morphological analysis, multiplex immunolabelling, immune‐gold labelling, and correlative light and electron microscopy workflow applicable for single cells, tissue and small organisms. This versatility makes AT attractive not only for basic research but as a diagnostic tool with a simplified routine. [ABSTRACT FROM AUTHOR]

Caenorhabditis elegans is a valuable model to study organ, tissue, and cell-type responses to external cues. However, the nematode comprises multiple syncytial tissues with spatial coordinates corresponding to distinct nuclear transcriptomes. Here, we present a single-nucleus RNA sequencing (snRNA-seq) protocol that aims to overcome difficulties encountered with single-cell RNA sequencing in C. elegans. We describe steps for isolating C. elegans nuclei for downstream applications including snRNA-seq applied to the context of alcohol exposure. For complete details on the use and execution of this protocol, please refer to Truong et al. (2023).1.

We report a technique to generate a murine model of lung metastases by selectively injecting tumor cells into the right heart ventricle under ultrasound guidance. First, we describe cell preparation and reference animal preparation as previously described. We then detail the technique using a previously described 3D-printed instrument stabilization device. Finally, we describe tumor growth surveillance using bioluminescent imaging. For complete details on the use and execution of this protocol, please refer to Labora et al.1.

Genetic approaches are limited in the dinoflagellate family, Symbiodiniaceae, causing a bottleneck in the discovery of useful mutants toward the goal of preventing future coral bleaching events. In this protocol, we demonstrate the application of UV exposure, coupled with downstream phenotypic screening and mutant isolation, to form a UV mutagenesis pipeline. This pipeline provides an avenue to generate Symbiodiniaceae mutants to help link genotype to phenotype, as well as address previously unanswered questions surrounding relationships with host organisms, like coral. For complete details on the use and execution of this protocol, please refer to Jinkerson et al. (2022).1.

Chimeric mouse models have recently been developed to study human microglia in vivo. However, widespread engraftment of donor microglia within the adult brain has been challenging. Here, we present a protocol to introduce the G795A point mutation using CRISPR-Cas9 into the CSF1R locus of human pluripotent stem cells. We also describe an optimized microglial differentiation technique for transplantation into newborn or adult recipients. We then detail pharmacological paradigms to achieve widespread and near-complete engraftment of human microglia. For complete details on the use and execution of this protocol, please refer to Chadarevian et al. (2023).1.

Model organisms are at the centre of progress in biology but attributing them an excessive representational power and concentrating on a limited group of them, although efficient for research, can have negative consequences, mainly of epistemic nature. Here, I argue that model organisms are exploratory models with a perspectival modelling function, and that a deflated representational power is needed for their proper use. In support of this argument, I will analyse developmental biology as a case study. Firstly, I show that model organisms in developmental biology are not selected because of their representational capabilities, but mainly based on practical criteria. Secondly, I defend that the epistemic organization of developmental biology around questions fosters exploration and perspectival modelling and I propose that developmental biology is a ‘model organism situated knowledge’. Lastly, I use the study of the mechanisms of cell fate acquisition during early embryonic development in C. elegans and mice as a case study to illustrate how a plurality of model organisms allows exploration and perspectival modelling. The use of model organisms for exploration and perspectival modelling, with a limited representational power, should allow more adequate inferences about human embryonic development and encourage the introduction of more model organisms for a comprehensive navigation of the space of possibilities. [ABSTRACT FROM AUTHOR]

The availability of compostable plastic bags has increased greatly in the past few years, as it is perceived that this type of bags will be degraded after disposal. However, there are some knowledge gaps regarding the potential effects on the soil ecosystems. We assessed the rate of degradation of samples of four different types of commercial compostable bags in vermicomposting systems with the earthworm species Eisenia andrei. We also evaluated the biological response of E. andrei (survival and reproduction) to microplastics (MPs) from fragments of the plastic bags (Lepidium sativum L.) exposed to micronized plastic (L. sativum seedling emergence was not significantly affected; however, earthworm reproduction was affected, suggesting that although compostable, some of the formulations may potentially be toxic to soil fauna.

The availability of compostable plastic bags has increased greatly in the past few years, as it is perceived that this type of bags will be degraded after disposal. However, there are some knowledge gaps regarding the potential effects on the soil ecosystems. We assessed the rate of degradation of samples of four different types of commercial compostable bags in vermicomposting systems with the earthworm species Eisenia andrei. We also evaluated the biological response of E. andrei (survival and reproduction) to microplastics (MPs) from fragments of the plastic bags (<2000 µm) and assessed seedling emergence in common garden cress (Lepidium sativum L.) exposed to micronized plastic (<250 µm) and the respective leachate, following OECD and ISO guidelines, respectively. The rate of degradation differed significantly depending on the type of plastic rather than the substrate in the vermicomposting system. This finding suggests that the degradation process is more dependent on the microbial community colonizing the different plastic types than on earthworm activity. Regarding the biological response of the soil system, L. sativum seedling emergence was not significantly affected; however, earthworm reproduction was affected, suggesting that although compostable, some of the formulations may potentially be toxic to soil fauna. [ABSTRACT FROM AUTHOR]

While model organisms have had many historians, this article places studies of humans, and particularly our development, in the politics of species choice. Human embryos, investigated directly rather than via animal surrogates, have gone through cycles of attention and neglect. In the past 60 years they moved from the sidelines to center stage. Research was resuscitated in anatomy, launched in reproductive biomedicine, molecular genetics, and stem-cell science, and made attractive in developmental biology. I explain this surge of interest in terms of rivalry with models and reliance on them. The greater involvement of medicine in human reproduction, especially through in vitro fertilization, gave access to fresh sources of material that fed critiques of extrapolation from mice and met demands for clinical relevance or "translation." Yet much of the revival depended on models. Supply infrastructures and digital standards, including biobanks and virtual atlases, emulated community resources for model organisms. Novel culture, imaging, molecular, and postgenomic methods were perfected on less precious samples. Toing and froing from the mouse affirmed the necessity of the exemplary mammal and its insufficiency justified inquiries into humans. Another kind of model—organoids and embryo-like structures derived from stem cells—enabled experiments that encouraged the organization of a new field, human developmental biology. Research on humans has competed with and counted on models. [ABSTRACT FROM AUTHOR]

Adolescence is a period of increased exploration and novelty-seeking, which includes new social behaviors, as well as drug experimentation, often spurred on by peer pressure. This is unfortunate, as the immature state of the adolescent brain makes it particularly susceptible to the negative developmental impact of drug use. During adolescence, dopamine terminals, which have migrated from the ventral tegmental area, pause in the nucleus accumbens, before segregating by either forming local connections or growing towards the prefrontal cortex (PFC). This developmentally late and lengthy process renders adolescent dopamine axon pathfinding vulnerable to disruption by substance use. Indeed, exposure to stimulant drugs in adolescent male mice, but not females, triggers dopamine axons to mistarget the nucleus accumbens and to grow ectopically to the PFC. Some evidence suggests that at this novel site, the functional organization of the ectopic dopamine axons mirrors that of the intended target. The structural rewiring dysregulates local synaptic connectivity, leading to poor impulse control ability, deficits of which are a core symptom of substance-use disorders. In the present commentary, we argue that different substances of abuse induce dopamine mistargeting events with the off-target trajectory prescribed by the type of drug, leading to psychiatric outcomes later in life. [ABSTRACT FROM AUTHOR]

Objective: Angiogenesis, defined as the formation of new blood vessels from pre-existing ones, plays a vital role in both physiological and pathological conditions. Understanding agents that can influence angiogenesis is crucial in managing diseases where angiogenesis is dysregulated. This study focuses on sevoflurane, a commonly used inhalation anesthetic, whose effects on angiogenesis and embryonic development are not well understood. Our objective was to assess the impact of sevoflurane exposure on angiogenesis using the ex-ovo chick chorioallantoic membrane model. Methods: In this model, fertilized chicken eggs were exposed to sevoflurane at concentrations of 2% and 4%, for varying durations of 1, 2, and 4 hours. The embryos were then divided into control and experimental groups for quantitative angiogenesis assessment using Image J software, followed by statistical analysis with one-way analysis of variance. Results: The results indicate that sevoflurane exposure has a dose-dependent positive effect on angiogenesis, with significant increases in vascular density observed in embryos exposed to both concentrations compared to the control group. Additionally, the length of exposure was found to further enhance these angiogenic effects. Conclusion: Despite the dose and duration-dependent impact of sevoflurane on angiogenesis, the existing literature presents mixed findings, highlighting the need for additional research to elucidate sevoflurane’s role in angiogenesis. This is particularly important for understanding its implications in various medical conditions, such as cancer, wound healing, and fetal development. Future investigations into sevoflurane’s effects on placental angiogenesis could also provide valuable insights into its potential consequences on intrauterine growth. [ABSTRACT FROM AUTHOR]

Aim: Patterns of individual variation are key to testing hypotheses about the mechanisms underlying biogeographic patterns. If species distributions are determined by environmental constraints, then populations near range margins may have reduced performance and be adapted to harsher environments. Model organisms are potentially important systems for biogeographical studies, given the available range‐wide natural history collections, and the importance of providing biogeographical context to their genetic and phenotypic diversity. Location: Global. Taxon: Arabidopsis thaliana ('Arabidopsis'). Methods: We fit occurrence records to climate data, and then projected the distribution of Arabidopsis under last glacial maximum, current and future climates. We confronted model predictions with individual performance measured on 2194 herbarium specimens, and we asked whether predicted suitability was associated with life history and genomic variation measured on ~900 natural accessions. Results: The most important climate variables constraining the Arabidopsis distribution were winter cold in northern and high elevation regions and summer heat in southern regions. Herbarium specimens from regions with lower habitat suitability in both northern and southern regions were smaller, supporting the hypothesis that the distribution of Arabidopsis is constrained by climate‐associated factors. Climate anomalies partly explained interannual variation in herbarium specimen size, but these did not closely correspond to local limiting factors identified in the distribution model. Late‐flowering genotypes were absent from the lowest suitability regions, suggesting slower life histories are only viable closer to the centre of the realized niche. We identified glacial refugia farther north than previously recognized, as well as refugia concordant with previous population genetic findings. Lower latitude populations, known to be genetically distinct, are most threatened by future climate change. The recently colonized range of Arabidopsis was well‐predicted by our native‐range model applied to certain regions but not others, suggesting it has colonized novel climates. Main Conclusions: Integration of distribution models with performance data from vast natural history collections is a route forward for testing biogeographical hypotheses about species distributions and their relationship with evolutionary fitness across large scales. [ABSTRACT FROM AUTHOR]

Here, we present a protocol to generate a murine model of liver metastasis by directly injecting tumor cells into the portal vein under ultrasound guidance. We describe steps for animal and cell preparation and two techniques for injecting tumor cells. One technique is freehand, while the other technique is device-assisted using a 3D-printed prototype device. Finally, we describe tumor surveillance with bioluminescent imaging.

Summary: Helicobacter-induced gastric cancer progresses very slowly, even in animal models, making it difficult to study. Here, we present a protocol to establish an accelerated murine model for Helicobacter-induced gastric cancer. We describe steps for infecting mice with Helicobacter felis, harvesting gastric tissue, assessing disease severity by histopathologic scoring, and performing gene expression studies with RT-qPCR and RNA sequencing. The accelerated model shows rapid progression of the disease, with gastric precancerous lesions developing within 6 months post-infection with Helicobacter.For complete details on the use and execution of this protocol, please refer to Bali et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: In cancer research, murine models play a crucial role as highly valuable preclinical tools. Here, we present a protocol to generate a murine model of glioblastoma through the direct intracranial injection of tumor cells. We describe steps for cell culture, intracranial implantation, and standard-of-care treatments. We then detail procedures for monitoring tumor growth using bioluminescent imaging.For complete details on the use and execution of this protocol, please refer to Pelizzari-Raymundo et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Applying Opal multiplex immunofluorescence (OMI) to characterize intestinal tissues of genetically engineered mouse models provides an excellent tool for studying complex processes. However, detecting appropriate signals from multiple target molecules is challenging. Here, we present a protocol to characterize mouse intestinal epithelial cell lineage using OMI. We describe steps for processing small intestine and colonic mouse tissues and designing and optimizing panels for OMI in mouse intestinal tissues. We then detail procedures for performing a quantitative evaluation of acquired images.For complete details on the use and execution of this protocol, please refer to Kinoshita et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Gene editing of human pluripotent stem cells is a promising approach for developing targeted gene therapies for metabolic diseases. Here, we present a protocol for generating a CRISPR-Cas12a gene knockout of protein tyrosine phosphatases in human embryonic stem cells. We describe steps for differentiating the edited clones into pancreatic islet-like spheroids rich in β-like cells. We then detail procedures for implanting these spheroids under the murine kidney capsule for in vivo maturation. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Isolation of amplifiable genomic DNA is a prerequisite for the implementation of PCR-based techniques. Here we present a protocol for isolating the genomic DNA from a variety of wild yeast species. This can be completed in approximately 1 h and does not require sophisticated laboratory equipment. We describe steps for growing yeast cells, genomic data extraction, and downstream assay for amplification of specific sequences from the genomic DNA. We then detail procedures for gel electrophoresis and analysis of the results.For complete details on the use and execution of this protocol, please refer to Kristjuhan et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Cancer cachexia mouse models are needed to recapitulate the clinical features of patients with cachexia. Here, we present a protocol for the establishment and evaluation of cancer cachexia mouse models. We delineate the steps in preparing tumor cells for inoculation and surgical procedures. After the establishment of these mouse models, we describe essential techniques to assess cancer cachexia, including grip strength evaluation, tissue collection, and the calculation of cross-sectional areas of muscle tissue.For complete details on the use and execution of this protocol, please refer to Liu et al.,1 Yang et al.,2 Shi et al.,3 and Zhou et al.4 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Generating a transgene with a reporter inserted into the genome helps us study endogenous gene expression patterns in model organisms. Here, using Drosophila melanogaster, we present a protocol for generating a P2A-Gal4 insertion through CRISPR-Cas9-mediated homology recombination. We describe the design strategy, steps for constructing the injection plasmids, and the fly-cross scheme for screening the transformants from the G0 generation. This protocol can also be applied to introduce mutations or various genetic tools into the fly genome.For complete details on the use and execution of this protocol, please refer to Li et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Metabolic disease complications pose a significant health risk due to their early development, making their diagnosis and radical therapy a considerable challenge. Here, we present a protocol for producing a rat model of non-obese prediabetes characterized by hyperinsulinemia, normoglycemia, and normal body weight. We describe steps for inducing the model in Sprague-Dawley (SD) male and ovariectomized female rats by free feeding on a mild hypercaloric diet. This protocol offers a potential model of metabolically unhealthy lean individuals.For complete details on the use and execution of this protocol, please refer to Elkhatib et al.1 and Dwaib et al.2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Gastrointestinal helminth infection occurs within a diverse microbiome, complicating the interpretation of whether effects are caused by the parasite versus the microbial community. Here, we present a protocol for deriving sterile larvae of the murine helminth, Heligmosomoides polygyrus bakeri (H. polygyrus), providing experimental control of the microbiome. We describe steps for sterilizing with a bleach solution and developing into infectious larvae using E. coli. We then detail procedures for removing bacterial contaminants before harvesting to ensure the generation of germ-free larvae. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Bacillus Calmette-Guérin (BCG), the only licensed tuberculosis vaccine, provides non-specific protection against non-tuberculosis diseases that is mediated by trained immunity, a functional reprogramming mediated by innate immune memory. Here, we present a protocol for analyzing BCG-induced trained immunity in murine bone marrow-derived macrophages (BMDMs). We describe steps for preparing BCG single bacterial suspensions, isolating BMDM cells, and the training process. This protocol can assist researchers to conveniently utilize BMDM cells to study trained immunity.For complete details on the use and execution of this protocol, please refer to Xu et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Spatial transcriptomics enables a single-cell resolution view of gene expression patterns in tissues, providing insight into their biological functions. However, applying this approach to the skin presents inherent challenges. Here, we present a protocol for preparing mammalian skin samples encompassing hair follicles for spatial transcriptomics. We describe steps for sample preparation, embedding, acquisition of frozen slices, RNA quality control, tissue mounting, fixation, staining, and imaging. We then detail procedures for permeabilization, reverse transcription, and cDNA collection.For complete details on the use and execution of this protocol, please refer to Chen et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Many types of neurons exhibit a daily rhythm of intrinsic excitability. Here, we present a protocol for assessing circadian regulation of dentate granule cell excitability using a mouse model for conditional knockout of the molecular clock protein BMAL1. We describe steps for obtaining healthy oblique horizontal slices that contain the hippocampus and measuring intrinsic excitability and synaptic potentials by combining whole-cell patch-clamp recordings and perforant-path electric stimulation. We then detail procedures for validating single-cell genetic deletion of Bmal1 by immunohistochemistry.For complete details on the use and execution of this protocol, please refer to Gonzalez et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Tardigrades are microscopic organisms with exceptional resilience to environmental extremes. Most protocols to visualize the internal anatomy of tardigrades rely on fixation, hampering our understanding of dynamic changes to organelles and other subcellular components. Here, we provide protocols for staining live tardigrade adults and other postembryonic stages, facilitating real-time visualization of structures including lipid droplets, mitochondria, lysosomes, and DNA. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Midbrain organoids provide an innovative cellular source for transplantation therapies of neurodegenerative diseases. Here, we present a protocol for midbrain organoid-derived cell transplantation into a Parkinson’s disease mouse model. We describe steps for midbrain organoid generation, single-cell suspension preparation, and cell transplantation. This approach is valuable for studying the efficacy of midbrain organoids as a potential cellular source for restoring motor function.For complete details on the use and execution of this protocol, please refer to Fu et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Here, we present a large-scale FLAG immunoprecipitation protocol to isolate large protein complexes driving DNA replication at replicating chromatin assembled in Xenopus laevis egg extract. We describe how to prepare demembranated sperm nuclei (DNA) and low-speed supernatant egg extract (LSS) and present detailed procedures for sample preparation and application onto grids for negative stain electron microscopy (NS-EM) and cryoelectron microscopy (cryo-EM).For complete details on the use and execution of this protocol, please refer to Cvetkovic et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Recapitulating spontaneous metastasis in preclinical models is crucial for understanding mechanisms underlying cancer progression and testing effective therapeutic interventions. We present a protocol for establishing and characterizing the spontaneous metastasis model in mice. We describe steps for generating primary tumors, tumor resection, monitoring metastatic dissemination, and evaluating metastatic burden using histological and imaging techniques. This protocol provides a valuable tool for studying metastasis in vivo and testing therapeutic strategies aimed at preventing or targeting metastatic diseases.For complete details on the use and execution of this protocol, please refer to Liu et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: In vivo models of brain pathology are crucial for studying neurological diseases. Here, we present a protocol to induce a pathological condition in a mouse brain area by local injection of neurotoxic stimulus. We describe steps for preparing reagents, stereotaxic injection procedures to induce neurodegeneration in the hippocampus, and preparation of brain sections to examine the induced model. This protocol is useful for studying how local pathology affects other brain areas and neighbor cells and its functional consequences in behavior.For complete details on the use and execution of this protocol, please refer to Zhang et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: The stop signal is produced in response to negative experiences at the food source and inhibits honey bee (Apis mellifera) waggle dancing. Here, we present a protocol for measuring the effects of an inhibitory signal associated with danger on honey bee dopamine levels. We describe steps for observing honey bee colonies, training them with artificial nectar, and simulating hornet attacks. We then detail procedures for recording waggle dancing and stop signals and measuring brain dopamine levels during different treatments.For complete details on the use and execution of this protocol, please refer to Dong et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Bacterial infections are the primary cause of pathogenic sepsis. An uropathogenic E. coli (UPEC) model of monomicrobial sepsis represents a useful tool for interrogating the host immune response to this pathogen. Here, we present a protocol for inducing monomicrobial sepsis in mice using UPEC. We describe steps for preparing the bacteria, delivering UPEC into mice, and monitoring the mice post-infection. We then detail procedures for measuring cytokine response and detecting immune cell subsets using flow cytometry.For complete details on the use and execution of this protocol, please refer to Martin et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Variable-rate delivery of intravenous drugs is difficult to achieve with a tethered infusion system in a freely moving animal. Here, we present a protocol for continuous intravenous delivery of oxytocin in pregnant rats and mice. We describe steps for using an implantable, preprogrammed, microprocessor-controlled infusion pump connected to the jugular vein to induce labor. This protocol can be adapted to a variety of experimental paradigms in non-pregnant animals where precise intravenous pharmacological manipulation is desired.For complete details on the use and execution of this protocol, please refer to Giri et al.1,2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Here, we present a protocol for the quantitative assessment of rat and mouse cardiomyocyte proliferation both in vitro and in vivo. For the in vivo approach, we describe steps for the isolation of neonatal rat cardiomyocytes and the employment of various indicators to quantify cell proliferation. We then detail in vivo procedures that incorporate comprehensive assays and a genetic lineage tracing strategy to evaluate endogenous cardiomyocyte proliferation. This protocol can be modified to investigate other mammalian cardiomyocyte proliferation.For complete details on the use and execution of this protocol, please refer to Ji et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: During aging and in retinal degenerative diseases, vulnerable retinal pigment epithelial (RPE) cells are subject to mitochondrial dysfunction, creating a need for accessibility to tools which can facilitate assessment of the ocular posterior pole bioenergetics. Here, we present a protocol for quantifying mitochondrial respiration in the posterior eye cup (RPE-choroid-sclera) of young and old mice. We describe steps for eye cup dissection, optimization of tissue size, drug concentrations, and cycle conditions using the XF Cell Mito Stress Test. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Mosquito behavioral assays are an important component in vector research and control tool development. Here, we present a protocol for rearing Anopheles mosquitoes, performing host-seeking behavioral bioassays, and collecting 3D flight tracks in a large wind tunnel. We describe steps for setting up host-seeking landing assays, both as a non-choice and as a dual-choice assay, and analyzing flight tracks. This protocol can be applied in the research of several behavioral traits, including nectar seeking, resting, mating, and oviposition behavior.For complete details on the use and execution of this protocol, please refer to Carnaghi et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: The fish disease model facilitates our understanding of disease dynamics, risk assessment for disease outbreaks, the response of the gut immune system, and the maintenance of ecosystem health. Here, we present a protocol for studying gut immunity modulation by infecting Lepidocephalichthys guntea, a loach fish, with Aeromonas hydrophila. We describe steps for performing intra-peritoneal injection on fish and a bath challenge. We detail procedures for conducting periodic population calculations during the infection phase to corroborate Aeromonas hydrophila invasion in the gut.For complete details on the use and execution of this protocol, please refer to Basak and Chakraborty.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Periodontal ligament cells (PDLCs) and macrophages in bone marrow cells have been widely used to investigate novel therapeutic agents to treat periodontitis. Here, we present a protocol for collecting primary mouse PDLCs and bone marrow cells. We detail steps for culturing and differentiation for both cell types and review data analysis for in vitro experiments using primary PDLCs and bone marrow cells. This protocol can be used to explore the impact of novel therapeutic agents using in vitro experiments.For complete details on the use and execution of this protocol, please refer to Sirisereephap et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: A gene-rescue experiment under a mutant background is essential to clarify gene function and the resulting biological potential in vivo. Here, we present a protocol for determining the change in interferon response by microinjecting plasmids into one-cell-stage zebrafish embryos. We describe steps for comparing the resistance potential to virus infection in wild-type and knockout zebrafish larvae following plasmid microinjection. We then detail how to link the enhanced interferon immunity to the improved resistance in knockout zebrafish larvae by gene-rescue experiments.For complete details on the use and execution of this protocol, please refer to Qu et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Humanized mice, defined as mice with human immune systems, have become an emerging model to study human hematopoiesis, infectious disease, and cancer. Here, we describe the techniques to generate humanized NSGF6 mice using adult human CD34+ hematopoietic stem and progenitor cells (HSPCs). We describe steps for constructing and monitoring the engraftment of humanized mice. We then detail procedures for tissue processing and immunophenotyping by flow cytometry to evaluate the multilineage hematopoietic differentiation.For complete details on the use and execution of this protocol, please refer to Yu et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Tissue-resident macrophages (TRMs) constitute the first line of defense against infection in all organs and perform organ-specific functions during tissue homeostasis. Here, we present a protocol for long-term monocultures of murine macrophages from different adult organs, including the brain, liver, peritoneal cavity, and lung. We describe steps for tissue preparation and the use of a combination of organotypic conditions to maintain a TRM-like identity in vitro, resulting in an ideal screening platform for a wide range of assays and readouts.For complete details on the use and execution of this protocol, please refer to Aktories et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: The anti-inflammatory activity of a phytocompound (oleuropein [OLP]) in the lipopolysaccharide (LPS)-mimicked macrophage model of inflammation demonstrates the importance of PI3K-Akt1 signaling in establishing “immune homeostasis.” Here, we present a protocol for the cultivation of in vitro cultures of P. falciparum for carrying out drug sensitivity assays. We describe steps for parasite synchronization, drug treatment, DNA isolation, and starvation-induced autophagy. This protocol provides insights into autophagy and parasite tolerance to drug pressure.For complete details on the use and execution of this protocol, please refer to Sharma et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.