Cell Biology

Daphnia magna is a well-established model organism in ecotoxicology, environmental monitoring, and genetics due to its sensitivity to pollutants, its pivotal role in freshwater ecosystems, and its well-characterized genome. Despite its extensive use in these fields, there is a notable lack of established protocols for developing primary cell culture systems and conducting transgenic studies in Daphnia spp. This study addresses these gaps by optimizing a medium and standardizing protocols for primary cell culture and transgenic experiments in D. magna. Primary cell cultures were established from both D. magna embryos and whole organisms, with medium optimization verified using XTT assay. Cell viability was sustained for over two months using a modified Schneider’s insect medium enriched with FBS, glucose, MEM vitamin mix, and selenium. DNA replication and cell proliferation were confirmed through BrdU labeling. Both mechanical and enzymatic passaging methods were compared, resulting in 20% and 10% cell attachment, respectively. For transgenic applications, this study successfully standardized plasmid-mediated lipofection and baculovirus-mediated transduction, achieving success rates of 52% and 45%. These findings represent a pioneering effort in D. magna embryonic cell culture, offering a reliable in vitro platform for future biological research, including ecotoxicological and epigenetic investigations. The established protocols and optimized cell culture medium have significant implications for advancing crustacean cell line research and transgenic model development, enhancing our understanding of biological processes in controlled laboratory environments.

Animal infection models play significant roles in the study of bacterial pathogenic mechanisms and host–pathogen interactions, as well as in evaluating drug and vaccine efficacies. Chlamydia trachomatis is responsible for infections in various mucosal tissues, including the eyes and urogenital, respiratory, and gastrointestinal tracts. Chronic infections can result in severe consequences such as trachoma-induced blindness, ectopic pregnancy, and infertility. While intravaginal inoculation of C. muridarum mimics the natural route of sexual transmission between individuals, transcervical inoculation allows the organisms to directly infect endometrial epithelial cells without interference from host responses triggered by chlamydial contact or infection of vaginal and cervical cells. Therefore, in this study, we used mouse models to visualize pathologies in both the endometrium and oviduct following C. muridarum inoculation.

Cell cultures play a crucial role in neuroscience research, facilitating the elucidation of the complexities of cellular physiology and pathology. The relative simplicity in producing cultures and the accessibility to cells that the cultures provide, in contrast to in vivo settings, allow users to manipulate and monitor cells more easily at higher throughputs and lower costs. These are ideal for screening purposes and electrophysiological characterizations. Despite the prevalence of methodologies for producing brain cultures from various animal models, rodents in particular, approaches for culturing neurons (and glia) from birds are less established or completely absent as in the case of the Japanese quail model. Here, we present a unique culturing protocol for brain cells (e.g., neurons at different maturation levels, such as progenitor cells, excitatory and inhibitory neurons, microglia, and endothelial cells) from entire forebrains of Japanese quail embryos for high-throughput screening of viral vectors in vitro and other various purposes. Following dissection and digestion methods uniquely suited for avian brains, we tailored the growth media and culturing surface to allow the survival of quail brain cultures for more than three weeks in vitro.

Current ischemic models strive to replicate ischemia-mediated injury. However, they face challenges such as inadequate reproducibility, difficulties in translating rodent findings to humans, and ethical, financial, and practical constraints that limit the accuracy of extensive research. This study introduces a novel approach to inducing persistent ischemia in 3-day-old chicken embryos using endothelin-1. The protocol targets the right vitelline arteries, validated with Doppler blood flow imaging and molecular biology experiments. This innovative approach facilitates the exploration of oxidative stress, inflammatory responses, cellular death, and potential drug screening suitability utilizing a 3-day-old chicken embryo.

Anemia is a common and serious health problem, nearly universally diagnosed in preterm infants, and is associated with increased morbidity and mortality worldwide. Red blood cell (RBC) transfusion is a lifesaving and mainstay therapy; however, it has critical adverse effects. One consequence is necrotizing enterocolitis (NEC), an inflammatory bowel necrosis disease in preterm infants. The murine model of phlebotomy-induced anemia and RBC transfusion–associated NEC enables a detailed study of the molecular mechanisms underlying these morbidities and the evaluation of potential new therapeutic strategies. This protocol describes a detailed procedure for obtaining murine pups with phlebotomy-induced anemia and delivering an RBC transfusion that develops NEC.

Insects rely on the simple but effective innate immune system to combat infection. Cellular and humoral responses are interconnected and synergistic in insects’ innate immune system. Phagocytosis is one major cellular response. It is difficult to collect clean hemolymph from the small insect like pea aphid. Here, we provide a practicable method for small insects hemocyte phagocytosis assay by taking pea aphid as an example. Furthermore, we provide the protocols for pea aphid rearing and bacterial infection, which offer referential method for related research.

Controlled differentiation of embryonic stem cells is an essential tool in stem cell research. In this protocol, we describe a simple differentiation protocol involving the induction of embryoid body formation in mouse embryonic stem cells (mESC) using hanging droplets, followed by differentiation into a neuronal lineage.

The early embryo of Drosophila melanogaster exists as a rapidly dividing syncytium of nuclei that are transcriptionally silent. Maternally deposited factors are required to awaken the genome and assist in the transition from maternal to zygotic control of development. Because many of these essential factors are maternally deposited and the early nuclear divisions are so rapid, it has been difficult to assess the functional role of transcription factors at discrete points in early embryonic development. To address this issue, we have developed an optogenetic system that can rapidly and reversibly inactivate transcription factors with nuclear-cycle resolution. The temporal precision enabled by this technique will allow a mechanistic understanding of how transcription factors function together to control genome activation and patterning in the early embryo and is likely broadly applicable to factors throughout embryogenesis.

Mitochondrial function and dysfunction are at the core of aging and involved in many age-dependent diseases. Rate of oxygen consumption is a measure of mitochondrial function and energy production rate. The nematode Caenorhabditis elegans (C. elegans) offers an opportunity to study “living” mitochondria without the need for mitochondrial extraction, purification and associated artifacts. Oxygen consumption rate (OCR) is traditionally measured using single-chamber Clark electrodes with or without the addition of metabolic modulators. More recently, multi-well oxygen electrodes with automated injection system have been developed to enable rapid measurement of OCR under different conditions. Here, we describe a detailed protocol that we have adapted from existing protocols to measure coupled and uncoupled mitochondrial respiration (with and without metabolic modulators) in live respiring nematodes using a Seahorse XFe96 extracellular flux analyzer. We present details on our protocol, including preparation of nematode culture, use of metabolic modulators, execution of Seahorse XF assay as well as post-experimental data analysis. As a reference, we provide results of a series of experiments in which the metabolic activity of N2 wild-type nematodes was compared to N2 nematode treated with paraquat, a compound that generates reactive oxygen species (ROS), thus causing oxidative damage and mitochondrial dysfunction. These data illustrate the kind of insights that can be obtained even using a low number of nematodes (10 animals only per well).

Eimeria vermiformis is a tissue specific, intracellular protozoan that infects the murine small intestinal epithelia, which has been widely used as a coccidian model to study mucosal immunology. This mouse infection model is valuable to investigate the mechanisms of host protection against primary and secondary infection in the small intestine. Here, we describe the generation of an E. vermiformis stock solution, preparation of sporulated E. vermiformis to infect mice and determination of oocysts burden. This protocol should help to establish a highly reproducible natural infection challenge model to study immunity in the small intestine. The information obtained from using this mouse model can reveal fundamental mechanisms of interaction between the pathogen and the immune response, e.g., provided by intraepithelial lymphocytes (IEL) at the basolateral site of epithelial cells but also a variety of other immune cell populations present in the gut.