Bio-protocol最新文献

The organ of Corti, located in the inner ear, is the primary organ responsible for animal hearing. Each hair cell has a V-shaped or U-shaped hair bundle composed of actin-filled stereocilia and a kinocilium supported by true transport microtubules. Damage to these structures due to noise exposure, drug toxicity, aging, or environmental factors can lead to hearing loss and other disorders. The challenge when examining auditory organs is their location within the bony labyrinth and their small and fragile nature. This protocol describes the dissection procedure for the cochlear organ, followed by confocal imaging of immunostained endogenous and fluorescent proteins. This approach can be used to understand hair cell physiology and the molecular mechanisms required for normal hearing. Key features • Protocol for the microdissection of the organ of Corti and suitable preparation for later immunostaining. • This technique involves the evaluation of mouse cochlea for planar-cell-polarity protein. • Quantitative and qualitative analysis of hair cell cilia in different dimensions.

Protein synthesis is by far the most energetically costly cellular process in rapidly dividing cells. Quantifying translating ribosomes in individual cells and their average mRNA transit rate is arduous. Quantitating assembled ribosomes in individual cells requires electron microscopy and does not indicate ribosome translation status. Measurement of average transit rates entails in vitro pulse-chase radiolabeling of isolated cells or ribosome profiling after ribosome runoff, which is expensive and extremely demanding technically. Here, we detail protocols based on ribosome-mediated nascent chain puromycylation, harringtonine to stall initiating ribosomes while allowing ribosome elongation to continue normally, and cycloheximide to freeze translating ribosomes in place. Each compound is delivered intravenously to mice in the appropriate order, and after ex vivo cell fixation and permeabilization, translating ribosome numbers and transit rates are measured by flow cytometry using a directly conjugated puromycin-specific antibody. Key features • Measure relative numbers of translating ribosomes in mixed single-cell preparations. • Quantitate relative in vivo ribosome transit rates in mixed single-cell preparations. • Detect ribosome stalling in vivo.

Cucumber (Cucumis sativus) trichomes play a critical role in resisting external biological and abiotic stresses. Glandular trichomes are particularly significant as they serve as sites for the synthesis and secretion of secondary metabolites, while non-glandular trichomes are pivotal for determining the appearance quality of cucumbers. However, current methods for separating trichomes encounter challenges such as low efficiency and insufficient accuracy, limiting their applicability in multi-omics sequencing studies. This protocol introduces an efficient system designed for the precise separation of glandular and non-glandular trichomes from cucumber fruit. The process begins with the pre-cooling of sorbitol buffer or ethanol solution and the RNA-free treatment of laboratory supplies, followed by sterilization and pre-cooling. After filling glass bottles with pre-cooling buffer and glass beads, cucumber ovaries are then placed in the glass bottles and the trichome is harvested by bead-beating method. The separation process involves sequential filtration through various steel sieves and centrifugation to separate trichomes. The separated trichomes obtained from this method are well-suited for subsequent multi-omics sequencing analyses. This protocol achieved high precision in separating glandular and non-glandular trichomes, significantly enhancing the efficiency of separation and sample collection processes. This advancement not only addresses existing limitations but also facilitates comprehensive studies aimed at exploring the genetic and biochemical diversity present within cucumber trichomes, thereby opening avenues for broader agricultural and biological research applications. Key features • Use cucumber fruits on the day of flowering. • Pre-cooling and RNA-free treatment ensure supply quality and purity. • Efficiently separate glandular and non-glandular trichomes. • Trichome samples are suitable for multi-omics sequencing analysis.

The bone is a highly dynamic organ that undergoes continuous remodeling through an intricate balance of bone formation and degradation. Hyperactivation of the bone-degrading cells, the osteoclasts (OCs), occurs in disease conditions and hormonal changes in females, resulting in osteoporosis, a disease characterized by altered microarchitecture of the bone tissue, and increased bone fragility. Thus, building robust assays to quantify OC resorptive activity to examine the molecular mechanisms underlying bone degradation is critical. Here, we establish an in vitro model to investigate the effect of estrogen withdrawal on OCs derived from the mouse macrophage RAW 264.7 cell line in a bone biomimetic microenvironment. This simple and robust model can also be adapted to examine the effect of drugs and genetic factors influencing OC resorptive activity in addition to being compatible with fluorescent imaging. Key features • A robust in vitro protocol that allows molecular and functional studies of mature osteoclasts in response to estrogen and its withdrawal. • Generation of inorganic bone-mimetic substrates for culturing and examining osteoclast resorptive behavior. • This quantitative image-based approach is compatible with brightfield and fluorescence microscopy to assess osteoclast resorptive activity.

The human intestine plays a pivotal role in nutrient absorption and immune system regulation. Along the longitudinal axis, cell-type composition changes to meet the varying functional requirements. Therefore, our protocol focuses on the processing of the whole human intestine to facilitate the analysis of region-specific characteristics such as tissue architecture and changes in cell populations. We describe how to generate a biobank that can be used to isolate specific immune cell subtypes, generate organoid lines, and establish autologous immune cell-organoid co-cultures. Key features • Dissection and tissue analysis of whole human intestines. • Cryopreservation for biobank generation. • Optimized protocols for the isolation of epithelial and immune cells. • Autologous co-culture of organoids and lamina propria-derived immune cells.

Agrobacterium-mediated gene transformation method is a vital molecular biology technique employed to develop transgenic plants. Plants are genetically engineered to develop disease-free varieties, knock out unsettling traits for crop improvement, or incorporate an antigenic protein to make the plant a green factory for edible vaccines. The method's robustness was validated through successful transformations, demonstrating its effectiveness as a standard approach for researchers working in plant biotechnology. It enables the introduction of foreign DNA into plant genomes. Conventionally, plant genetic transformation has relied on time-consuming, costly, and technically demanding procedures, such as electroporation and chimeric viruses or biolistic methods, which usually yield variable transformation efficiencies. This study presents a simple and fail-safe protocol that involves a modified freeze-thaw and heat-shock concoction method. This approach involves a streamlined plasmid miniprep procedure to isolate high-quality plasmid DNA from Escherichia coli K12 strain, followed by a target-specific transfer into A. tumefaciens EHA105 strain. The optimized method minimizes DNA degradation and maximizes uptake by Agrobacterium cells, making it a reproducible and accessible protocol for various genetic engineering applications. The transformation efficiency is consistently high, enhancing plasmid uptake while maintaining cell viability, requiring minimal specialized equipment and reagents. The proposed protocol offers significant advantages, including simplicity, reliability, and cost-effectiveness, positioning it as a valuable alternative to traditional techniques in the field of plant biotechnology. Key features • Uses liquid nitrogen as a proxy for freezing. • Plasmid DNA from competent bacterial cells is extracted using a user-friendly high-copy isolation kit. • A maximum of five consecutive days is sufficient to complete the procedures.

Mitochondrial cristae, formed by folding the mitochondrial inner membrane (IM), are essential for cellular energy supply. However, the observation of the IM is challenging due to the limitations in spatiotemporal resolution offered by conventional microscopy and the absence of suitable in vitro probes specifically targeting the IM. Here, we describe a detailed imaging protocol for the mitochondrial inner membrane using the Si-rhodamine dye HBmito Crimson, which has excellent photophysical properties, to label live cells for imaging via stimulated emission depletion (STED) microscopy. This allows for STED imaging over more than 500 frames (approximately one hour), with a spatial resolution of 40 nm, enabling the observation of cristae dynamics during various mitochondrial processes. The protocol includes detailed steps for cell staining, image acquisition, image processing, and resolution analysis. Utilizing the superior resolution of STED microscopy, the structure and complex dynamic changes of cristae can be visualized. Key features • The protocol is designed to visualize mitochondrial cristae in living cells using STED microscopy. • The protocol enables nanoscale observation of dynamic mitochondrial cristae. • Real-time observation of mitochondrial morphological changes, fusion, and fission events.

Candida auris, labeled an urgent threat by the CDC, shows significant resilience to treatments and disinfectants via biofilm formation, complicating treatment/disease management. The inconsistencies in biofilm architecture observed across studies hinder the understanding of its role in pathogenesis. Our novel in vitro technique cultivates C. auris biofilms on gelatin-coated coverslips, reliably producing multilayer biofilms with extracellular polymeric substances (EPS). This method, applicable to other Candida species like C. glabrata and C. albicans, is cost-effective and mimics the niche of biofilm formation. It is suitable for high-throughput drug screening and repurposing efforts, aiding in the development of new therapeutics. Our technique represents a significant advancement in Candida biofilm research, addressing the need for consistent, reproducible biofilm models. We detail a step-by-step procedure for creating a substratum for biofilm growth and measuring biofilm thickness using confocal laser scanning microscopy (CLSM) and ultrastructure by scanning electron microscopy (SEM). This method provides consistent outcomes across various Candida species. Key features • The biofilm formed on gelatin surfaces mimics host conditions, replicating the multilayered structure and EPS, offering a more accurate model for studying C. auris biofilms. • This method is highly reproducible and suitable for drug screening and biofilm analysis through three-dimensional (3D) reconstruction. • This in vitro technique aids in studying biofilm formation, related virulence properties, and drug tolerance of C. auris and other Candida species. • The simple, cost-effective technique is ideal for screening novel inhibitors and repurposed drug libraries, facilitating the design/identification of new therapeutics against Candida species.

Neurons are highly polarized cells, with axons that may innervate distant target regions. In the brain, basal forebrain cholinergic neurons (BFCNs) possess extensive axons that project to several target regions such as the cortex, hippocampus, and amygdala, and may be exposed to a specific microenvironment in their axon targets that may have retrograde effects on neuronal health. Interestingly, BFCNs express the pan-neurotrophin receptor p75NTR throughout life while also concomitantly co-expressing all Trk receptors, making them capable of responding to both mature and precursor neurotrophins to promote survival or apoptosis, respectively. Levels of these trophic factors may be modulated in the BFCN axon or soma microenvironment under neurodegenerative conditions such as seizure and brain injury. In this protocol, BFCNs are established in microfluidic devices for compartmental culture, with the aim of studying the effects of axon- or soma-specific stimulation of BFCNs for an in vitro representation of distal axon vs. soma environments as seen in vivo. This study further establishes a novel method of tracing and imaging live BFCNs exposed to stimuli in their distal axons with the aim of assessing retrograde cell death. The in vitro compartmental culture system of BFCNs that allows live imaging may be applied to investigate various effects of axon- or soma-specific stimuli that affect BFCN health, maintenance, and death, to model events that occur in the context of brain injury and neurodegenerative disorders. Key features • Separation of axons and soma of basal forebrain primary neurons in vitro using microfluidic chambers. • Compartmental/localized treatment of axons or somas of BFCNs. • Live imaging of retrogradely labeled BFCNs to assess cell death.

Histological techniques to study muscle are crucial for assessing skeletal muscle health. To preserve tissue morphology, samples are usually fixed in formaldehyde or cryopreserved immediately after excision from the body. Freezing samples in liquid nitrogen, using isopentane as a mediator for efficient cooling, preserves the tissue in its natural state. However, this method is highly susceptible to freeze-fracture artifacts, which alter or destroy tissue architecture. Isopentane is most commonly used in a semi-frozen/liquid state that is visually assessed by the experimenter, which can pose a challenge when freezing multiple tissues at a time or maintaining a consistent temperature. Furthermore, tissue size is also a confounding factor; depending on the size, freezing times can vary. In this study, we compare two different options for using isopentane while cryopreserving tissue. We also present an easy and reproducible method of freezing the soleus tissue of mice using frozen isopentane. This method decreased the occurrence of freeze-fractures by an order of magnitude, to ~4%, whereas the traditional method of cryopreservation resulted in ~56% freeze-fracturing. Key features • A uniform and highly reproducible protocol for freezing any tissue that is prone to freeze-fracture. • Removes the need to maintain a mixed state of isopentane. • Optimized cryopreservation method for the soleus muscle of mice. • Allows for prevention of peripheral freeze-fracture in tissue, which is the most susceptible region to freeze-fracture damage. Graphical overview.