علوم آزمایشگاهی پزشکی
مقالهها، منابع و پژوهشهای تازه حوزه علوم آزمایشگاهی پزشکی
ورود به زیرشاخهنقشه موضوعی
جستوجوی دقیقعلوم آزمایشگاهی و تشخیص
تشخیص آزمایشگاهی، تصویربرداری و فناوریهای تشخیصی
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مقالهها، منابع و پژوهشهای تازه حوزه هماتولوژی آزمایشگاهی
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مقالهها، منابع و پژوهشهای تازه حوزه میکروبشناسی آزمایشگاهی
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ورود به زیرشاخهرادیولوژی و تصویربرداری
مقالهها، منابع و پژوهشهای تازه حوزه رادیولوژی و تصویربرداری
ورود به زیرشاخهپزشکی هستهای
مقالهها، منابع و پژوهشهای تازه حوزه پزشکی هستهای
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ورود به زیرشاخهتازهترین رکوردها
شواهد علوم آزمایشگاهی و تشخیص
PubMed2027
A Defined Strategy for Multi-Lineage Differentiation of Gastric Organoids.
In the gastric glands, adult stem cells differentiate into multiple epithelial lineages, including pit mucous cells, acid-secreting parietal cells, and enzyme-producing chief cells. Efficient and controllable lineage specification from adult gastric stem cells is essential for modeling gastric homeostasis and disease in vitro. Organoids are a good model for investigating cell differentiation and organ formation. However, methods to achieve appropriate differentiation of multiple gastric lineages are inefficient. Here, we provide a detailed stepwise protocol for the differentiation of functional cells in human or mouse gastric organoids through targeted modulation of key signaling pathways. The workflow enables reproducible generation of distinct gastric epithelial cell types and provides a tractable platform to dissect signaling requirements. This platform facilitates downstream mechanistic exploration and enhances the translational utility of gastric organoids for disease modeling and therapeutic development.
PubMed2027
A Microscopy Toolkit for Root Studies in B. distachyon.
The study of plant organogenesis presents significant challenges due to the small number of cells involved in its initial stages. Observing the initial cell divisions becomes increasingly challenging when transitioning from Arabidopsis thaliana (Arabidopsis) to other species. Lateral root (LR) initiation is an essential process for enhancing a plant's ability to access water and nutrients. In most cases, LR formation starts in the pericycle of the parent root, giving rise to a new meristem. Understanding the mechanisms coordinating LR development is important for improving plant resilience to biotic and abiotic stresses. The prediction of the precise timing and location of LR initiation along the root axis remains challenging, even in Arabidopsis. This is magnified when attempting to observe LR development in crops. Brachypodium distachyon (Brachypodium) has emerged as a versatile model for cereal crops. However, studying Brachypodium LR development requires a revision of protocols and methodologies to be applied from seedling growth to root imaging. Here, we present protocols for seed preparation, in vitro growth, and tissue clearing for Brachypodium. Whereas ClearSee appeared to be unsuitable for rendering Brachypodium root tissues transparent after a reasonable incubation period, our modified DEEP-Clear method resulted in improved efficiency in tissue clearing and is compatible with major fluorescent proteins and dyes. Finally, we introduce a simple and straightforward approach to locally synchronize LR development. These tools and methodologies are crucial for advancing our knowledge of plant root system architecture, transitioning from a model plant to agronomically important species.
PubMed2027
A Standardized Protocol for Generating iPSC-Derived Human Microglia for Functional Genomic Assays.
Human induced pluripotent stem cell (iPSC)-derived microglia (iMG) provide an in vitro experimental system for studying human microglial biology, neuroinflammation, and genetic risk mechanisms associated with neurological disease. This chapter describes a standardized, scalable, and reproducible protocol for the differentiation of human iPSCs into functional microglia-like cells, with particular emphasis on applications in transcriptional and epigenomic network analysis. The protocol supports high-viability floating iMG production, compatibility with pooled CRISPR perturbation approaches, and downstream multiomic profiling, including single-cell RNA sequencing, chromatin accessibility assays, and proteomics. Detailed procedures are provided for iPSC maintenance, hematopoietic progenitor cell generation, microglial maturation, functional genomics integration, and quality control.
PubMed2027
AA and Pregnancy.
AA is a crucial fatty acid in pregnancy, especially for the growth and development of the fetus and its central nervous system (CNS). As already discussed in the previous chapters, AA regulates inflammation, immune response, and cell signaling. AA has a modulatory action on gene expression, serves as a mechanotransducer, and regulates cell membrane fluidity. By regulating cell membrane fluidity, AA modulates the expression and binding of several receptors located on the cell membrane. Eicosanoids derived from AA have both beneficial and harmful effects on pregnancy. For instance, (i) AA metabolites such as 14,15-EET and 15-HETE are associated with gestational hypertension and preeclampsia, (ii) AA in association with DHA is crucial for the fetal nervous system and neurocognitive development of the newborn, and (iii) for the growth and development of the fetus, especially during the third trimester. LA and AA are involved in human fertility and in the pathobiology of gestational diabetes.
PubMed2027
AA in Hypertension and Preeclampsia.
Human essential hypertension is driven by a network of interacting nutritional, metabolic, inflammatory, and endothelial mechanisms in which AA and other PUFAs play a critical role. Beyond excess sodium intake, population data supports the role of inadequate calcium, potassium, and magnesium intake and low antioxidant vitamin status in shaping blood pressure regulation. These nutrients influence vascular smooth muscle tone and act as cofactors that support Δ6 and Δ5 desaturase activities, thereby governing tissue availability of AA, EPA, and DHA and downstream formation of vasodilator and antiplatelet mediators (e.g., PGE1, prostacyclin), as well as inflammation-resolving lipid mediators (lipoxins, resolvins, protectins, maresins, and nitrolipids).Endothelial dysfunction characterized by reduced endothelial nitric oxide (eNO) bioavailability and increased oxidative stress seems to play an important role in the pathobiology of HTN. High salt intake, asymmetric dimethylarginine (ADMA), and activation of NAD(P)H oxidase and angiotensin II signaling promote superoxide generation that quenches NO and shifts the vascular balance toward vasoconstriction. Clinical and experimental observations indicate that antihypertensive therapies can partly restore NO and antioxidant defenses, while dietary patterns rich in n-3 fatty acids (particularly DHA) and balanced n-6/n-3 intake may lower blood pressure by suppressing thromboxane formation and enhancing vasoprotective, pro-resolving pathways.Preeclampsia can be considered as a model of reversible hypertension linked to oxidative stress and angiogenic imbalance (sFlt1, soluble endoglin), highlighting mechanistic overlap with essential hypertension. Hypertension is a low-grade systemic inflammatory condition whose origins may be present in the perinatal period through long-term programming of PUFA metabolism and endothelial function.
PubMed2027
Alkaline Phosphatase Staining of Embryonic Stem Cells.
Alkaline phosphatase (AP) is a widely utilized histochemical marker for assessing the pluripotent state of embryonic stem (ES) cells. Mouse ES cells maintained in an undifferentiated state exhibit high levels of tissue-nonspecific alkaline phosphatase (TNAP) activity, which sharply declines upon differentiation. AP staining provides a rapid, cost-effective, and reliable method to evaluate ES cell colony morphology, confirm maintenance of pluripotency during routine culture, and monitor the efficiency of differentiation protocols. Here, we describe a detailed protocol for AP staining of mouse ES cells using a chromogenic azo dye coupling method with naphthol AS-BI phosphate substrate and Fast Red Violet LB diazonium salt. This technique produces a vibrant red-purple precipitate in undifferentiated ES cell colonies, while differentiated cells remain unstained, enabling clear visualization and documentation of pluripotency status. The protocol includes optimized fixation conditions that preserve AP enzymatic activity, streamlined staining procedures, and guidelines for image acquisition and interpretation.
PubMed2027
An Electroporation-Based Protocol for Ex Vivo Base Editing: From Design to Quantitative Assessment.
Base editing is a CRISPR variant approach that enables single-nucleotide conversions without generating double-strand breaks. Cytosine and adenine base editors mediate C•G to T•A and A•T to G•C transitions, respectively, by coupling a deaminase to a catalytically impaired Cas9, a modified nuclease that lacks DNA cleavage activity but retains DNA binding capability. By avoiding double-strand breaks, base editing limits reliance on unpredictable end-joining pathways and facilitates more precise outcomes. This chapter provides a practical protocol for base editing by electroporation in adherent and suspension stem cells. The workflow spans guide design aligned with the editor activity window, in vitro synthesis of mRNA-based editor components, and electroporation for transient delivery. We detail essential steps for cell handling and recovery, followed by standard readouts. Analytical endpoints focus on targeted sequencing to quantify base conversion and assess editing specificity, with guidance for basic interpretation of results. Troubleshooting notes address frequent pitfalls and practical remedies to optimize performance across cell types.
PubMed2027
An Integrated Workflow for Culturing and AI-Based Profiling of Mouse Intestinal Epithelium.
Two-dimensional (2D) culture systems are powerful in vitro tools that have revolutionized the study of intestinal stem cell biology. These culture systems derived from mouse intestinal and colonic epithelia enable the modeling of tissue architecture, barrier function, and disease mechanisms under physiologically relevant conditions. This protocol details methods for isolating and maintaining mouse intestinal crypts as 2D monolayers. Monolayers are established via enzymatic dissociation and seeding onto ECM-functionalized hydrogels. This protocol also describes a deep learning pipeline that performs virtual fluorescent staining of label-free phase-contrast images of live organoids. This protocol enables the reproducible generation and computational analysis of murine intestinal epithelial cultures for studies in epithelial biology, drug testing, and disease modeling. This integrated system is designed for facilitating high-throughput imaging, immunolabeling, and functional analysis, expanding its use in regenerative medicine, tissue engineering, and gastrointestinal research.
PubMed2027
Analysis of Cell Populations from Arbovirus-Infected Tissues by Flow Cytometry.
Arboviruses still represent a major challenge to public health in many parts of the world. Despite shared epidemiological characteristics, each arboviral disorder displays distinct cellular tropism and activation patterns, progressing to immunopathological outcomes that remain not fully elucidated. A deeper understanding of these processes requires methodologies capable of characterizing the often-complex cellular responses. In this context, flow cytometry emerges as a valuable tool, enabling the characterization of heterogeneous cell populations, the assessment of activation and proliferation markers, the quantification of inflammatory mediators, and the detection of cell death pathways. By integrating these parameters, flow cytometry can enhance our understanding of arbovirus pathogenesis, clarify their cellular tropism, and understand immune responses to infection. This methodological article outlines the main steps for applying the technique to different target tissues infected by chikungunya, dengue, yellow fever, and Zika viruses, emphasizing the most effective strategies.
PubMed2027
Analysis of Spatial Gene Expression in Chlamydia Using Fluorescent In Situ Hybridization.
The obligate intracellular bacterium Chlamydia employs a unique, asynchronous biphasic developmental cycle characterized by distinct morphological forms: the replicative reticulate body (RB), the infectious elementary body (EB), and an intermediate body (IB). The simultaneous presence of these phenotypically distinct cell types throughout infection complicates the study of gene expression regulation during development. Conventional population-level assays are inadequate for dissecting the regulatory mechanisms within this mixed population. This chapter describes a robust method utilizing hybridization chain reaction (HCR) RNA fluorescence in situ hybridization (FISH) in conjunction with dual-promoter reporter Chlamydia strains to determine cell-form-specific transcript expression. We leverage dual-reporter strains to provide visual identification of RBs and EBs. Probes targeting genes of interest are designed to interact with spectrally distinct fluorescent HCR amplifiers. This versatile, multiplexing system allows for precise spatial and temporal localization of specific mRNAs within identified Chlamydial cell forms, offering a powerful tool to overcome current limitations in Chlamydia research and elucidate the regulatory underpinnings of its complex life cycle.
PubMed2027
Animal-Free Intestinal Epithelial 2D "Organoid-Sheet" Cultures Using Invasin.
The intestinal epithelium consists of a single layer of polarized cells that serves as a crucial barrier against pathogens while mediating food digestion and nutrient absorption. The differentiated villus cells, important for the intestine's function, originate from rapidly dividing Lgr5+ stem cells located in the intestinal crypts. These stem cells can be expanded in vitro and form three-dimensional (3D) "mini-organs" known as intestinal organoids, which closely phenocopy the intestinal epithelium. The development and maintenance of these 3D organoids depend on a defined growth factor cocktail and on extracellular matrix (ECM) support to preserve apical-basal polarity - an essential feature for nutrient uptake, growth, and the barrier function. Matrigel or BME has been used as the standard ECM hydrogel scaffold for organoid culture. Recently, we identified the bacterial protein Invasin as a defined ECM substitute that can fully recapitulate Matrigel/BME function. Utilizing recombinant Invasin coated on transwells, we developed a two-dimensional (2D) intestinal monolayer culture system. The intestinal stem cells form a polarized, confluent epithelial sheet while maintaining stemness and differentiation capacity into functional epithelial cell types. Compared to traditional 3D cultures, this 2D system offers advantages such as improved imaging, easier access to apical and basal compartments, and compatibility with automation for high-throughput applications. Here, we present a detailed protocol for generating 2D intestinal monolayers using Invasin, starting from either fresh human intestinal tissue or from pre-established 3D organoids. These 2D Invasin-cultures recapitulate key aspects of intestinal epithelial biology and are amenable to a broad range of experimental manipulations, including studies on gene function, disease modeling, pathogen interaction, co-cultures, and cell fate determination. These defined 2D-Invasin "organoid-sheets" cultures support the realization of clinical applications using healthy and patient-derived intestinal epithelial cells.
PubMed2027
Assays of Antifungal Activity of an Antimicrobial Peptide Against Botrytis cinerea.
Antimicrobial peptides (AMPs) represent the first line of host immune defense against a broad spectrum of microorganisms. Whether naturally occurring or synthetically designed, these small peptides can potently inhibit microbial growth and remain stable under diverse conditions. Most AMPs act by directly disrupting fungal or bacterial membranes. In this study, we identified a glycine-rich peptide with strong antifungal activity against Botrytis cinerea. We further investigated its mode of action (MOA) and demonstrated its preventive antifungal activity in plants. Our work focuses primarily on the in vitro characterization of AMP features in the context of plant-Botrytis interactions.
PubMed2027
Assessing Sensitivity to Antifungal Plant Compounds or Agrochemicals.
Botrytis cinerea, a major fungal pathogen of crops, has been extensively studied for its resistance to antifungal plant metabolites and agrochemicals. Evaluating the sensitivity of B. cinerea to these compounds is crucial for understanding its pathogenicity and for developing effective plant protection strategies. This study describes two methods for antifungal sensitivity assessment: the spot assay, which visualizes fungal growth on agar medium supplemented with antifungal compounds, and the resazurin assay, which quantifies fungal metabolic activity in liquid culture. Using saponins as antifungal compounds, we demonstrate the utility of these methods on B. cinerea. These methods provide simple, reliable, and reproducible protocols to assess the sensitivity of antifungal compounds on B. cinerea.
PubMed2027
Assessing the Vector Competence for Chikungunya Virus in Mosquitoes.
Chikungunya virus (CHIKV) is a human pathogenic, mosquito-borne virus (arbovirus), which is causing epidemic outbreaks among human populations in Africa, Asia, Europe, and South- and Central America including the Caribbean. The development of novel approaches to prevent mosquito transmission of the virus in the field requires a detailed study of CHIKV interactions with its mosquito vectors in the laboratory. In this chapter, we describe how to prepare Aedes aegypti mosquitoes for the infection with freshly cultivated CHIKV. We also describe quantitative and qualitative viral detection assays in mosquitoes based on plaque assays and the amplification of saliva samples in cell culture.
PubMed2027
Automated Counting of Chikungunya Virus-Formed Plaques.
The cytopathic effect (CPE) is a key to understanding the pathogenicity of the chikungunya virus (CHIKV) and is useful for evaluating viral infectivity in vitro. The plaque assay, based on the CPE induced by viral infection, provides an infectious virus titer for test samples. Additionally, the assay is simple, quantitative, and cost-effective. Thus, the plaque assay remains the gold standard for determining the infectivity of CHIKV. However, manually counting of virus-induced plaques with the naked eye or under the microscope is often time-consuming and labor-intensive. Therefore, automated plaque-counting software would improve the consistency and efficiency of plaque counting. We recently developed plaQuest, a stand-alone Windows software that enables rapid, reliable plaque counting for CHIKV. In this chapter, we describe the basic procedure for detecting and counting the CHIKV-formed plaques in a 24-well plate.
PubMed2027
Automated Quantification and Morphometric Analysis of Pluripotent Stem Cell Colonies Using ColonyQuant.
Alkaline phosphatase staining is routinely used to evaluate the undifferentiated state of embryonic stem cells, yet quantitative interpretation of colony assays frequently depends on manual inspection and categorical scoring that introduce subjectivity and limit scalability. ColonyQuant provides a standardized, automated framework for objective analysis of alkaline phosphatase-stained colonies from conventional bright-field images. The software performs adaptive colony detection, per-colony intensity measurement, and extraction of eight geometric descriptors that collectively characterize colony size, compactness, symmetry, and boundary complexity. Feature tables generated by the workflow are structured to support statistical comparison across replicates and experimental conditions. Integrated analysis modules enable dimensionality reduction, supervised classification, and feature ranking, facilitating interpretation of phenotypic differences without requiring custom scripting. Visualization routines generate distribution plots, contour-density maps, multivariate embeddings, and representative shape mosaics to summarize population heterogeneity and morphological organization. Applied to pluripotent stem cell cultures subjected to chromatin perturbation, the platform detects coordinated changes in colony growth behavior and structural architecture that may not be evident through visual scoring alone. The protocol describes installation, configuration, batch image processing, quality control, hierarchical data aggregation, and downstream statistical analysis, providing a reproducible approach for transforming qualitative colony assays into quantitative, high-content phenotypic datasets suitable for stem cell research and screening applications.
PubMed2027
Build Heatmaps of Cell Frequencies and Marker Intensities for Correct Interpretations.
High-content cytometry is an important technique in clinical research, producing data that is rich and complex to analyze. The resulting clusters of cells must be profiled to determine their role and function and analyzed to identify changes in cell frequency or marker intensity. The heat map is the most suitable tool for presenting this information in a synthetic way. However, its construction must be adapted to the information and the objective. Here we describe the step-by-step construction of heat maps of marker intensity and cell frequency using Excel, Phantasus, or R, including the conversion of numbers to colors and the organization of rows and columns. Researchers who master these steps will be able to correctly interpret heat maps and derive the most benefit from high-content cytometry.
PubMed2027
Cell Biology of Botrytis: Live-Cell Imaging Techniques and Protein Localization Protocols.
Botrytis cinerea is a widely studied model organism for plant-fungal interactions. This chapter provides simple and easy protocols for live-cell imaging techniques and subcellular protein localization studies in B. cinerea in vitro. Using a simple microscopy setup, we describe methods for imaging spore germlings and hyphae and for studying protein dynamics in response to the antifungal compound α-tomatine. The protocols highlight the application of fluorescence microscopy to visualize sterol distribution and protein localization patterns, enabling deeper insights into fungal cell biology.
PubMed2027
Characteristic Analysis of the Resting-State fMRI Global Signal in Schizophrenia.
The global signal (GS) represents the broad variations in neural activity throughout the brain. Recent research has identified changes in the GS of functional magnetic resonance imaging (fMRI) in schizophrenia, challenging the traditional view of GS as mere noise that is typically discarded during data preprocessing. However, there has been no comprehensive characteristic analysis of GS in schizophrenia. This study recruited 100 patients with schizophrenia and 113 healthy subjects to perform resting-state fMRI. The averaged gray matter fMRI signal is denoted as GS. The GS topography was constructed by calculating Pearson correlation (i.e. GSCORR) between the GS and time series of each gray matter voxel. Furthermore, the relevance between GS topography and the function network features was constructed according to graph theory. Finally, we implemented an integrated analytical framework combining independent component analysis and multiple linear regression to quantify the contributions of resting-state brain networks to the spatiotemporal characteristics of the GS. The GSCORR of schizophrenia decreased in the bilateral insula and exhibited a significant negative association with disease duration. The increased GSCORR in the thalamus and default mode network (DMN) showed a positive correlation with the scale scores. We found that global properties are represented by GS topography in schizophrenia. In addition, the linear characterization results of GS showed that the component contribution decreased at the primary sensory network but increased at the high-order associated network. Our results further demonstrated that GS contains brain features associated with schizophrenia, which would help to understand the neural mechanisms underlying the psychopathological symptoms of schizophrenia.
PubMed2027
Chikungunya Virus Infection in Paraffin-Embedded Tissue: Analysis of Histological Alterations and Viral Detection by Immunohistochemistry.
Histopathological analysis of tissues infected with Chikungunya virus (CHIKV), including biopsy and autopsy specimens, can provide valuable insights into the pathogenesis of atypical and fatal cases. By examining tissue architecture and cellular alterations under the microscope, it is possible to identify patterns of injury, inflammation, and cellular degeneration. These morphological findings provide evidence of how the pathogen interacts with host cells and tissues. Moreover, immunohistochemistry may be performed in tissue sections for the detection of viral antigens. Importantly, this technique allows the characterization of diverse biologically relevant targets in tissue samples, such as distinct immune cell subsets, cytokines, and additional molecular markers. This is accomplished by the use of antibodies selected for their specificity toward the antigen of interest. In this chapter, we present a concise overview of how histological analysis and immunohistochemical approaches can enhance the understanding of CHIKV-associated pathological mechanisms.
PubMed2027
Chlamydia trachomatis Infection in a Microphysiologic Cell Culture Model.
Chlamydia trachomatis is an obligate intracellular bacterium that infects the columnar epithelium of the human endocervix. While conventional two-dimensional cell cultures and animal models have been instrumental in advancing our understanding of C. trachomatis biology, they are limited in capturing the multicellularity, architecture, and physiological microenvironment of the human cervix. This chapter describes the use of a three-dimensional (3D) microphysiologic model to study Chlamydia trachomatis infection. The model is inexpensively made without specialized equipment and is designed to recreate the epithelial-stromal interface. We outline procedures for coculturing cervical epithelial cells and fibroblasts, infecting epithelial cells with fluorescently labeled C. trachomatis, monitoring infection progression via fluorescent microscopy, and quantifying infectious progeny. The complete developmental cycle of C. trachomatis within this model provides a robust and accessible platform to investigate C. trachomatis-specific host-pathogen interactions, immune responses, and the influence of diverse physiological and environmental stimuli within a relevant cervical context.
PubMed2027
Clinical Flow Cytometric Testing in Chronic Lymphocytic Leukemia.
Flow cytometry is the cornerstone for establishing the diagnosis of chronic lymphocytic leukemia (CLL), owing to its characteristic and well-defined immunophenotype that enables accurate distinction from other leukemias and lymphomas. Beyond diagnosis, flow cytometry provides essential prognostic information and allows sensitive detection of minimal residual disease (MRD), a strong predictor of clinical outcome. CLL MRD assessment is increasingly used to guide risk stratification, therapeutic decision-making, and treatment duration in the era of targeted therapies and immunotherapies. This chapter reviews best practices for specimen collection, processing, staining, and data analysis and summarizes the principles of flow cytometric MRD assessment in CLL.
PubMed2027
Comprehensive Isolation Strategy for Intestinal Epithelial and Fibroblast Cells from Healthy and Tumor Human Tissues.
Organoids and fibroblasts derived from patient tissue serve as physiologically meaningful in vitro models to investigate tissue biology, diseases, and treatment responses. In this study, we present a robust protocol for the simultaneous isolation and long-term culture of patient-derived organoids (PDOs) and fibroblasts from both healthy and colorectal cancer samples. The workflow uses enzymatic dissociation followed by efficient separation into epithelial and stromal cell fractions. Organoids are embedded in Matrigel or Matrigel-Collagen I mixtures to support three-dimensional growth, whereas fibroblasts are maintained on conventional two-dimensional culture dishes. This dual-culture approach facilitates a broad range of downstream applications.
PubMed2027
Deep Insights Without Clearing: An Optimized Protocol for Patient-Derived Whole-Organoid Immunofluorescence Imaging.
Here, we present an optimized whole-organoid immunofluorescence staining protocol that preserves morphology, achieves uniform antibody penetration, and enables deep-tissue imaging without the need for tissue clearing. Organoids are three-dimensional, self-organizing structures that replicate many of the cellular and architectural features of their tissue of origin, making them powerful preclinical models for studying development, disease, and therapeutic responses. Most organoid research is performed on formalin-fixed, paraffin-embedded (FFPE) samples, in which organoids are sectioned across the Z-planes. While this method facilitates conventional histological analysis, it limits spatially resolved visualization of protein expression and tissue organization. The inherent density and abundant extracellular matrix of organoids present substantial barriers to reagent penetration and imaging depth, further constraining the ability to capture their three-dimensional architecture in full. Our new workflow includes steps for fixation, permeabilization, and antibody incubation and has been validated on intestinal organoids using markers for different membrane-associated proteins. By providing high-quality staining through the full organoid depth without clearing, this protocol streamlines sample preparation, reduces processing artifacts, and allows more rapid, accurate 3D mapping of cell types and structures in organoid-based research.
PubMed2027
Delivery of Targeting Constructs into Zygotes Using Electroporation.
Zygote electroporation (EP) is a widely used technique for the delivery of targeting constructs into the embryos and has become very popular in the last few years in transgenic facilities all over the world. Zygote electroporation is technically simple, as it does not require equipment for microinjection. Moreover, zygote electroporation is less invasive, resulting in higher survival rates in comparison to pronuclear microinjection, leading to a reduction in the number of animals needed. Another advantage of zygote electroporation is a higher number of zygotes that can be targeted simultaneously, which minimizes the time required for zygote manipulation. Zygote electroporation is easily combined with the use of the CRISPR-Cas9 system.
PubMed2027
Determination of Chikungunya Virus Infectious Titer Using Plaque Assays.
The plaque-forming assay is the gold-standard method for measuring infectious viral particles. In this technique, lytic viruses infect and destroy host cells, but their spread is restricted by a viscous overlay medium. As a result, new viral particles can only infect neighboring cells, leading to localized clear areas known as plaques, which become visible after staining the remaining living cells. The number of plaques reflects the number of infectious viral particles initially present in the sample and is reported as plaque-forming units (PFU) per sample volume. In this protocol, we describe step by step how to perform a plaque-forming assay to determine the concentration of chikungunya virus in a cell culture supernatant.
PubMed2027
EdU-Based Quantitative Analysis of Cell Proliferation in Arabidopsis Root Meristems.
The division of a mother cell into two daughter cells is a fundamental process in biology, involving the transfer of genomic information to the next generation of cells. In plant roots, cell divisions occur in the root apical meristem, a specialized tissue located at the plant's root tip. Cell proliferation rates in the root apical meristem shape root growth, thereby contributing to general development as well as the acclimatization to stressful environmental conditions through phenotypic plasticity. Here, we present a protocol for the microscopical analysis of cell proliferation in Arabidopsis thaliana root meristems by 5-ethynyl-2'-deoxyuridine (EdU) staining. EdU, a thymine-analog, is incorporated into newly synthesized DNA of proliferative cells during the S-phase of the cell cycle. Subsequently, EdU-containing nuclei can be labeled with a fluorophore and detected by confocal laser scanning microscopy. The combination of the EdU assay with cell wall staining enables the quantification of root meristem architecture parameters and cell division activity from microscopic images. Together, this can elucidate how root meristems respond to changes in environmental conditions or address fundamental questions of developmental plant biology.
PubMed2027
Electroporation for High-Efficiency Delivery of CRISPR to Hematopoietic Cells.
Recent advances in genome editing technologies have enabled transformative therapeutic strategies for hematological disorders. Efficient implementation of these approaches requires reliable delivery of genome editing components into primary hematopoietic stem and progenitor cells (HSPCs), which are particularly sensitive and resistant to conventional transfection methods. Electroporation has emerged as the most widely used strategy for ex vivo delivery of genome editing reagents into CD34⁺ hematopoietic cells. Genome editing tools can be delivered in multiple formats, including plasmid DNA, messenger RNA (mRNA), and ribonucleoprotein (RNP) complexes. In HSPCs, mRNA- and RNP-based approaches are generally better tolerated than plasmid DNA and allow transient expression with reduced cytotoxicity. In this chapter, we describe optimized protocols for electroporation-based delivery of CRISPR/Cas9 RNP complexes and mRNA-encoded base editors into HSPCs using the Amaxa™ 4D-Nucleofector™ system. Detailed procedures are provided for cell preparation, guide RNA design, assembly of Cas9 RNPs, in vitro transcription and purification of base editor mRNA, electroporation parameters, and assessment of editing efficiency. Emphasis is placed on maximizing editing performance while preserving stem cell viability and functionality.
PubMed2027
Enhancing the Functionality of Human Bone Marrow-Derived Mesenchymal Stromal Cells Using Signaling Modulators.
Bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) possess an inherent capacity to support hematopoiesis in vitro. As a result, they are often used as feeder layers for the ex vivo expansion of hematopoietic stem cells (HSCs) or coinfused with HSCs to improve the success of clinical transplants. However, MSCs isolated from non-hematopoietic tissues such as adipose or dental tissue may not be as effective as BM-MSCs in supporting hematopoiesis. Additionally, BM-MSCs tend to lose their functional ability after extended in vitro culture, which is necessary for producing sufficient cell numbers for clinical use. These limitations underscore the importance of pretreating MSCs with suitable signaling modulators to boost their functionality. Reliable in vitro assays play a crucial role in finding such signaling compounds and evaluating their effects on MSCs. Conventional assays, such as long-term-culture-initiating assay (LTC-IC assay) or colony-forming-unit assay (CFU assay), chemotactic migration assays (e.g., Transwell assay), etc., primarily evaluate the functional competence of HSCs and HSPCs, but provide little insight into the hematopoiesis-supportive capacity of MSCs. This chapter describes two such assays: one is designed to assess the hematopoietic stem/progenitor cell (HSPC)-supporting capacity of primed MSCs, and the other is specifically aimed at their ability to support the growth of primitive HSCs. These assays can serve as initial screens to test the impact, whether positive or negative, synergistic or antagonistic, of known signaling compounds, bioactive peptides, growth factors, and cytokines on MSC function or to discover new entities that could boost the functionality of MSCs.
PubMed2027
Experimental Evolution of Drug Resistance for Antibiotic Target Discovery in Chlamydia trachomatis.
The recent application of experimental and computational drug discovery workflows has identified hundreds of new molecules that display potent, and in some cases highly selective, activity against bacteria of the genus Chlamydia. The full value of these novel antichlamydial molecules, however, depends critically on our ability to determine their molecular targets and modes of action. A particularly powerful and widely applied strategy for identifying candidate targets involves selecting for bacterial mutants that have acquired resistance to a compound's inhibitory activity, followed by determining the mutations responsible for this resistance. Such mutations often pinpoint the compound's direct target or reveal cellular pathways that modulate target engagement. Here, we present a detailed, step-by-step protocol for applying this approach to the clinically important, human-pathogenic species Chlamydia trachomatis. The workflow includes: (1) determining compound potency by quantitative dose-response analysis, (2) generating a resistant C. trachomatis mutant through serial passaging under progressively increasing selective pressure, (3) confirming the resistance phenotype, and (4) isolating bacterial genomic DNA for whole-genome sequencing to identify resistance-associated mutations and facilitate downstream target discovery.