Jove-Journal of Visualized Experiments最新文献

Accurate assessment of Newcastle disease virus (NDV) antibody titers is crucial for effective poultry disease control and surveillance. This article introduces an optimized method for preparing 4 hemagglutination units (4-HAU) antigen solution, a key component of the hemagglutination inhibition assay (HI) used in NDV serological detection. Unlike conventional methods, which involve time-consuming and undefined back-titration and adjustment steps, this approach streamlines this process by accurately measuring the HA titer using an initial series of dilutions (1:3, 1:5, 1:7, and 1:9). We also provide a specific method for adjusting or reformulating based on the back-titration results, reducing the need for repeated back-titrations. In addition, we evaluated the effect of the accuracy of the 4-HAU antigen solution on serum HI titer and found that when the titer of the 4-HAU antigen was lower than 3, it resulted in the appearance of false-positive HI samples. By providing an accurate method and minimizing computational tasks, this approach increases test efficiency and reliability, contributing to improved disease surveillance and control in poultry populations.

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few protocols are available that allow an in vitro approximation of the in vivo situation. Here, we present a novel approach to observing the impact of human macrophages on the invasiveness of cancer cells, using tumor spheroids of H1299 non-small cell lung carcinoma cells embedded in a three-dimensional (3D) collagen I matrix. With this co-cultivation setup, we tested the impact of small interfering RNA (siRNA)-based depletion of regulatory factors in macrophages on the 3D invasion of cancer cells from the tumor spheroid compared to controls. This method allows the determination of different parameters, such as spheroid area or the number of invading cancer cells, and thus, to detect differences in cancer cell invasion. In this article, we present the respective setup, discuss the subsequent analysis, as well as the advantages and potential pitfalls of this method.

A multi-system approach that includes the continuous collection of electroencephalogram (EEG) and electrocardiogram (ECG) recordings facilitates a comprehensive assessment of electrical activity in the brain and heart, particularly leading up to and surrounding episodic events. Building on our previous protocol that facilitates the collection of intermittent EEG/ECG recordings from conscious restrained rabbits, we developed a protocol for the collection of continuous EEG/ECG recordings from unrestrained rabbits. This new method enables high-quality 24/7 recordings in the housing cage, which captures the normal range of physiological states and enables the rabbits to move, eat, drink, and sleep freely. It enables comprehensive assessments of the prevalence, incidence, and susceptibility to EEG/ECG abnormalities, seizures, arrhythmias, and sudden death. This procedure involves the surgical placement of subdermal electrodes and the design and manufacturing of a robust wiring system that is impervious to damage by the rabbits. The surgical procedure includes two incisions and the sub-dermal tunneling of nine electrodes and wires that exit through a custom-made port. The external connector attaches to a wire harness that includes an electrical swivel, which allows for free range of rabbit motion throughout its cage. Additionally, the wires are enclosed in a metal sheath suspended by a retractor cable from the ceiling of the rabbit's cage. The wires are then connected to an amplifier/digitizer for the acquisition of high-quality continuous EEG/ECG recordings.

The rise of multi-drug-resistant infections and the lack of new antibiotic classes have renewed interest in alternative therapies like photodynamic inactivation of bacteria (aPDI). This process involves the administration of a photosensitizer (PS) activated by a suitable visible light source, producing exacerbated levels of reactive oxygen species (ROS) that damage crucial cellular biomolecules, ultimately causing bacterial cell death. It is crucial to create standardized, easy-to-use, and reproducible initial tests to assess and compare the effectiveness of light-induced phototoxicity of potential photosensitizers. This study introduces a simple and efficient in vitro technique for assessing photodynamic activity against planktonic bacterial cells. By employing a 96-well microplate format along with a large LED panel, the system facilitates the systematic evaluation of various compounds. Such a configuration allows for high-throughput screening of potential photosensitizers in a controlled and consistent environment, simplifying the process of identifying promising candidates for further development. This flexible platform serves as an important step in advancing the development of innovative photodynamic therapies for managing antibiotic-resistant infections.

Evaluating respiratory drive presents challenges due to the obtrusiveness and impracticality of current methods like functional magnetic resonance imaging (fMRI). Electromyography (EMG) offers a surrogate measure of respiratory drive to the muscles, allowing the determination of both the magnitude and timing of muscle activation. The magnitude reflects the level of muscle activation, while the timing indicates the onset and offset of muscle activity relative to specific events, such as inspiratory flow and activation of other muscles. These metrics are critical for understanding respiratory coordination and control, especially under varying loads or in the presence of respiratory pathophysiology. This study outlines a protocol for acquiring and analyzing respiratory muscle EMG signals in healthy adults and patients with respiratory health conditions. Ethical approval was obtained for the studies, which included participant preparation, electrode placement, signal acquisition, preprocessing, and postprocessing. Key steps involve cleaning the skin, locating muscles via palpation and ultrasound, and applying electrodes to minimize electrocardiography (ECG) contamination. Data is acquired at a high sampling rate and gain, with synchronized ECG and respiratory flow recordings. Preprocessing includes filtering and transforming the EMG signal, while postprocessing involves calculating onset and offset differences relative to the inspiratory flow. Representative data from a healthy male participant performing incremental inspiratory threshold loading (ITL) illustrate the protocol's application. Results showed earlier activation and prolonged duration of extradiaphragmatic muscles under higher loads, correlating with increased EMG magnitude. This protocol facilitates a detailed assessment of respiratory muscle activation, providing insights into both normal and pathophysiologic motor control strategies.

Adipose tissue is primarily composed of mature, lipid-laden adipocytes by volume. These postmitotic cells play a critical role in energy storage and mobilization, thermoregulation, and the secretion of endocrine factors. The expansion of white adipose tissue due to caloric imbalance results in both the enlargement of existing adipocytes and the generation of additional adipocytes from adipocyte progenitor cells. Obesity-driven changes to white adipose tissue, including those affecting adipocytes, are associated with numerous comorbidities, such as type 2 diabetes and 13 types of cancer. A significant barrier to studying how adipocytes contribute to disease is the inability to readily isolate and culture mature adipocytes. This article describes a protocol to isolate murine lean and obese adipocytes from the subcutaneous and visceral fat depots of male and female C57BL/6 mice. The protocol details how isolated primary adipocytes can be cultured in a membrane adipocyte aggregate system for up to 2 weeks, facilitating their functional analysis in co-culture experiments, lipolysis assays, or through the collection of conditioned media containing adipocyte-secreted factors. Additionally, the protocol outlines methods for culturing adipose tissue explants in basement membrane matrix domes and imaging primary isolated adipocytes. Importantly, this approach can be integrated with existing protocols for the isolation of adipose tissue-resident adipocyte progenitor cells using fluorescence-activated cell sorting (FACS). Together, these protocols provide researchers with tools to functionally study adipocytes, adipocyte progenitor cells, and whole adipose tissue from lean and obese, male and female mice.

For over a century, Treponema pallidum subsp. pallidum, the spiral-shaped bacterium that causes syphilis, could only be propagated by inoculation and harvest of the organisms from rabbit testes. In 2018, we described a method to continuously cultivate T. pallidumin vitro. This system utilizes co-culture with rabbit epithelial cells (Sf1Ep cells) in a serum-containing tissue culture medium called TpCM-2. The T. pallidum doubling time in culture is similar to that estimated to occur during natural infection (about 33-45 h). The organism can be cultured continuously with a standard passage time of 1 week in a low oxygen (1.5%) environment at 34 °C. This article contains the protocols for culturing T. pallidum, methods for growing and maintaining the required tissue culture cells, and the technique for generating isogenic strains by limiting dilution. The ability to grow T. pallidum in vitro provides new experimental avenues to study and understand this enigmatic organism.

Bacterial-fungal interactions (BFIs) play an integral role in shaping microbial community composition, biogeochemical functions, spatial dynamics, and microbial dispersal. Mycelial networks created by filamentous fungi or other filamentous microorganisms (e.g., Oomycetes) act as 'fungal highways' that can be utilized by bacteria for transport throughout heterogeneous environments, greatly facilitating their mobility and granting them access to regions that may be challenging or impossible to reach on their own (e.g., due to air pockets within the soil). Several devices and experimental protocols have been created to study these fungal highways, including fungal highway columns. The fungal highway column designed by our group can be used for a variety of in situ or in vitro applications, as well as with diverse environmental and host-associated sample types. Herein, we describe the methods for performing experiments with these columns, including designing, printing, sterilizing, and preparing the devices. The options for analyzing data obtained from the use of these devices are also discussed here, and troubleshooting advice regarding potential pitfalls associated with experiments using fungal highway columns is offered. These devices can be used to gain a more comprehensive understanding of the diversity, mechanisms, and dynamics of fungal highway BFIs to provide valuable insights into the structural and functional dynamics within complex environments (e.g., soils) and across diverse habitats in which bacteria and fungi co-exist.

Nanogels consisting of crosslinked-polymeric nanoparticles have been developed for the delivery of numerous chemical and biological therapeutics, owing to their versatile bottom-up synthesis and biocompatibility. While various methods have been employed for nanogel synthesis to date, very few have achieved it without the use of harsh organic solvents or high temperatures that can damage the integrity of the biological payload. In contrast, the methodology presented here accomplishes the synthesis of sub-100 nm sized, protein-loaded nanogels using mild reaction conditions. Here, we present a method for the non-covalent encapsulation of protein-based payloads within nano-gels that were synthesized using an aqueous-based, single-step, crosslinking copolymerization technique. In this technique, we initially electrostatically bind a protein-based payload to a cationic quaternary ammonium monomer and simultaneously cross-link and co-polymerize it using ammonium persulfate and N,N,N',N'-tetramethylethylenediamine to form nanogels that entrap the protein payload. The size and polydispersity index of the nanogels is determined using dynamic light scattering (DLS), while the surface morphology is assessed by transmission electron microscopy (TEM). The mass of protein entrapped within nanogels is determined by calculating the encapsulation efficiency. Furthermore, the controlled-release ability of the nanogels via the gradual degradation of redox-responsive structural elements is also assessed in bioreduction assays. We provide examples of nanoparticle optimization data to demonstrate all caveats of nanogel synthesis and characterization using this technique. In general, uniformly sized nanogels were obtained with an average size of 57 nm and a polydispersity index value of 0.093. A high encapsulation efficiency of 76% was achieved. Furthermore, the nanogels exhibited controlled release of up to 86% of the encapsulated protein by gradual degradation of novel redox-responsive components in the presence of glutathione over 48 h.

Cryo-electron tomography (cryo-ET) is a powerful technique for visualizing the ultrastructure of cells in three dimensions (3D) at nanometer resolution. However, the manual segmentation of cellular components in cryo-ET data remains a significant bottleneck due to its complexity and time-consuming nature. In this work, we present a novel segmentation workflow that integrates advanced virtual reality (VR) software to enhance both the efficiency and accuracy of segmenting cryo-ET datasets. This workflow leverages an immersive VR tool with intuitive 3D interaction, enabling users to navigate and annotate complex cellular structures in a more natural and interactive environment. To evaluate the effectiveness of the workflow, we applied it to the segmentation of mitochondria in retinal pigment epithelium (RPE1) cells. Mitochondria, essential for cellular energy production and signaling, exhibit dynamic morphological changes, making them an ideal test sample. The VR software facilitated precise delineation of mitochondrial membranes and internal structures, enabling downstream analysis of the segmented membrane structures. We demonstrate that this VR-based segmentation workflow significantly improves the user experience while maintaining accurate segmentation of intricate cellular structures in cryo-ET data. This approach holds promise for broad applications in structural cell biology and science education, offering a transformative tool for researchers engaged in detailed cellular analysis.