Methods in molecular biology (Clifton, N.J.)Nanshan Zhong, Fan Hong, Yiran Luo, Ye-Guang Chen
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.
Methods in molecular biology (Clifton, N.J.)Jason Ngo, Emily Lee, Marta Olah, Falak Sher
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.
Methods in molecular biology (Clifton, N.J.)Benjamin L Kidder
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.
Methods in molecular biology (Clifton, N.J.)Irene Peña-Gutierrez, Davide Mazzeo, Alex Bassons-Bascuñana, Sibtain Haider, Laura Garcia-Garcia, Beatriz Olalla-Sastre, Paula Río, Sergio López-Manzaneda, Man…
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.
Methods in molecular biology (Clifton, N.J.)Farah Qaderi, U Kaicheng, Yeganeh Farsi, Junhan Zhao, Nima Saeidi
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.
Methods in molecular biology (Clifton, N.J.)Jenniffer Ramos Martins, Viviane Lima Batista, Celso M Queiroz-Junior, Mauro Martins Teixeira, Vivian Vasconcelos Costa
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.
Methods in molecular biology (Clifton, N.J.)Nicole A Grieshaber, Scott S Grieshaber
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.
Methods in molecular biology (Clifton, N.J.)Joost J A P M Wijnakker, Hans Clevers
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.
Methods in molecular biology (Clifton, N.J.)Min Zhang, Po Hu, Hailing Jin
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.
Methods in molecular biology (Clifton, N.J.)H M Suraj, André Fleissner, Jan A L van Kan
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.
Methods in molecular biology (Clifton, N.J.)Alexander W E Franz
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.
Methods in molecular biology (Clifton, N.J.)Akino Emi, Youichi Suzuki
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.
Methods in molecular biology (Clifton, N.J.)Benjamin L Kidder
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.
Methods in molecular biology (Clifton, N.J.)Eugénie Lohmann, Laurent Gorvel, Samuel Granjeaud
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.
Methods in molecular biology (Clifton, N.J.)Natália G Salomão, Kíssila Rabelo
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.
Methods in molecular biology (Clifton, N.J.)David J Delgado Diaz, Katherine M Nelson, Vonetta L Edwards, Ian J Glomski, Filipa Ribeiro, Patrik M Bavoil, Alison K Criss, Jacques Ravel, Jason P Gleghorn, I…
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.
Methods in molecular biology (Clifton, N.J.)Nadia Nasir, Maryalice Stetler-Stevenson, Hao-Wei Wang
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.
Methods in molecular biology (Clifton, N.J.)Samna Sagadevan, Paloma Ordóñez-Morán
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.
Methods in molecular biology (Clifton, N.J.)Laura Gull, Mireia Sueca-Comes, Ana Pires
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.
Methods in molecular biology (Clifton, N.J.)Michaela Vaskovicova, Vojtech Dolejs, Pavla Dostalova, Csilla Sustr Michalikova, Jan Prochazka, Radislav Sedlacek
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.
Methods in molecular biology (Clifton, N.J.)Sebastiano Volpe, Claudia Filomatori
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.
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.
Methods in molecular biology (Clifton, N.J.)Nikoletta Y Papaioannou, Panayiota L Papasavva, Petros Patsali, Carsten W Lederer
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.
Methods in molecular biology (Clifton, N.J.)Vaijayanti Kale
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.
Methods in molecular biology (Clifton, N.J.)Aakriti Singh, Barbara S Sixt
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.
Methods in molecular biology (Clifton, N.J.)Dana Delgado, Jolene L Cardinali, Joseph A DiGiuseppe
Immunophenotyping by flow cytometry is an important component in the diagnostic evaluation of patients with acute lymphoblastic leukemia. This technique further permits the detection of minimal residual disease after therapy, a robust prognostic factor that may guide individualized treatment. We describe here laboratory methods for both the initial characterization of lymphoblasts at diagnosis and the detection of rare leukemic lymphoblasts after treatment. In addition to antibody combinations suitable for diagnosis and detection of minimal residual disease, we describe procedures for peripheral blood and bone marrow sample preparation, procedures for labeling of cell-surface and intracellular proteins with fluorochrome-conjugated antibodies, and approaches to analysis of immunophenotypic data, including those obtained in patients following CD19-targeted therapies.
Botrytis cinerea, a notorious pathogenic fungus, leads to postharvest decay in horticultural crops and substantial economic losses, while the pathogenicity of B. cinerea is closely related to its structural characteristics and cellular homeostasis. Among the currently available techniques, fluorescence and ultrastructural observations serve as crucial tools for investigating subcellular structures and cellular events during B. cinerea growth. These techniques enable the monitoring of cell structural integrity, such as variations in the cell membrane and cell wall, as well as intracellular changes, such as reactive oxygen species bursts and cell apoptosis. The results at cytological levels may not only facilitate close-up dissection of B. cinerea cells, but also provide important cues to uncover the inhibitory mechanism of exogenous treatments. This chapter introduces fluorescence staining and observation, as well as the preparation of ultrathin sections of B. cinerea conidia, aiming to provide methodological guidance for cytological examinations of B. cinerea.
Methods in molecular biology (Clifton, N.J.)Alessio Terenzi, Elena Salvi, Riccardo Di Mambro
In plant roots, the diversity of tissues along the radial axis and of developmental stages along the longitudinal axis implies a highly dynamic and heterogeneous gene expression. Many master regulators of gene expression exhibit distinctive temporal expression patterns or are restricted to specific cell types, making whole-root molecular analyses insufficient to resolve their roles in developmental and physiological processes. Here, we report a protocol for fluorescence-activated cell sorting (FACS)-coupled RNA sequencing to analyze, reconstruct, and ultimately dissect the gene regulatory networks (GRNs) involved in root development and physiology, particularly well-suited for genes that are expressed transiently or in rare or restricted cell types. The method relies on selecting an appropriate cell-type-specific reporter line, which enables the isolation of labeled cells with high specificity and minimal contamination. Furthermore, we discuss the relative advantages of FACS-coupled RNA-seq compared with alternative gene-expression methodologies, including quantitative PCR (qPCR), bulk root RNA-seq, single-cell RNA-seq (scRNA-seq), and spatial transcriptomics, considering sensitivity, resolution, input requirements, and cost.
Methods in molecular biology (Clifton, N.J.)Kaho Washizu, Akira Kunitomi
The Sendai virus (SeV) vector system offers an efficient, nonintegrating approach to reprogram somatic cells into either naive or primed human induced pluripotent stem cells (iPSCs). Here, we describe a protocol to generate transgene-free naive iPSCs from human dermal fibroblasts (HDFs) and peripheral blood mononuclear cells (PBMCs) using a modified, temperature-sensitive SeV system. The method leverages LMYC in place of cMYC and an optional H1FOO-DD factor to enhance efficiency and uniformity, and employs a controlled temperature shift to facilitate vector clearance.
Methods in molecular biology (Clifton, N.J.)Janet Staats, Anagha Divekar, J Philip McCoy, Holden T Maecker
"Gating" refers to the selection of successive sub-populations of cells for analysis in flow cytometry. While automated analysis algorithms have seen a greatly increased use, there is still heavy reliance on manual gating, based on expert knowledge of cell characteristics. This can lead to variability in how gates are applied, even between individuals experienced in the field. For clinical assays, guidelines often exist, and software may automate the gating process. But there are no comprehensive guidelines across the various types of immunological assays performed in research settings using flow cytometry. Here, we attempt to provide such guidelines, focused on the most general and pervasive types of gates, why they are important, and what recommendations can be made regarding their use. We do so through the display of example data, collected by academic, government, and industry representatives. These guidelines should be of value to both novice and experienced flow cytometrists analyzing a wide variety of immunological assays.
Methods in molecular biology (Clifton, N.J.)Kim Han
Immunophenotyping of human peripheral blood mononuclear cells (PBMCs) is a critical technique for understanding immune responses in both health and disease. Traditionally, this process relies on the use of liquid antibody cocktails, which are time-consuming to prepare, prone to laboratory error, and have limited shelf lives. Recent advancements in lyophilization of antibodies and other reagents have addressed these challenges by enhancing reagent stability, extending shelf life, and simplifying transport and storage. Dried antibody cocktails have been shown to perform comparably to liquid cocktails in flow cytometry assays while reducing preparation time and minimizing handling errors. This review focuses on the use of lyophilized reagents for high-dimensional immunophenotyping of human PBMCs, discussing their advantages in large-scale studies, global health initiatives, and clinical research. By streamlining immune profiling workflows, lyophilized reagents have the potential to improve accessibility and facilitate the adoption of immunophenotyping in resource-limited settings.
Spectral cytometry has rapidly evolved over the past two decades. The development of complete spectral analysis using the flow cytometer as a spectrometer has been actualized due to advances in optics, detectors, and electronics that have made single-cell flow spectroscopy a reality. Polychromatic flow cytometry is a complex process, including the design of antibody panels and instrument compensation. Spectral cytometry has contributed significantly to effectively dealing with the complexity of polychromatic flow cytometry. It has substantial potential to improve primary and clinical research. Here, we provide an overview of the methodology to develop a 15-color human polychromatic flow cytometry panel using spectral flow cytometry.
Methods in molecular biology (Clifton, N.J.)Emiko Desvaux, Amazigh Mokhtari
Recent advances in multiomics technologies have revolutionized the study of immune-driven diseases by enabling high-dimensional, single-cell resolution analyses. This chapter provides a practical guide for integrating CyTOF (mass cytometry) and single-cell RNA sequencing (scRNA-seq) data to address key challenges in this field. The integration of these modalities allows for consistent and reproducible cell type annotation, the transfer of annotations to assist in characterizing difficult-to-identify populations, and the transcriptional characterization of rare and heterogeneous subpopulations. Using tools such as OMIQ and R, the chapter outlines workflows for preprocessing, normalization, and scaling of CyTOF data, as well as dimensionality reduction and clustering techniques. The integration process involves creating Seurat objects, identifying common features, and using anchor-based methods to link CyTOF and scRNA-seq datasets. The chapter also discusses the use of multimodal deep learning techniques for rare subpopulation detection and emphasizes the importance of reproducibility and standardization in multiomics integration. By leveraging these methodologies, researchers can gain deeper insights into cellular heterogeneity and function, ultimately enhancing the understanding of immune-driven diseases. The chapter concludes by addressing integration challenges and proposing future directions for improving model interpretability and capturing nonlinear molecular interactions.
Methods in molecular biology (Clifton, N.J.)Adriana Martinez-Silgado, Lulu Huang, Joep Beumer, Hans Clevers
The small intestine plays a crucial role in nutrient uptake and metabolism regulation while maintaining a barrier that protects against the harsh environment of the lumen. These different tasks are carried out by a multitude of cell types, continuously generated by the resident stem cell population, and whose specification is under tight regulatory control. Organoids are self-organizing 3D structures derived from tissue-resident stem cells, which retain the ability to differentiate into all other epithelial lineages. Protocols to enrich for the various cell subtypes in the intestine have greatly advanced in recent years. Here, we provide an updated overview of these protocols, which direct human small intestinal organoids differentiation into various mature cell lineages, recapitulating the in vivo gut.
Here, we present detailed protocols for histological staining and imaging of root cross sections of the fern Ceratopteris richardii, providing high-resolution visualization of cellular and tissue structures essential for understanding root development, nutrient and water uptake, and responses to environmental cues. This chapter describes the preparation of root samples, fixation, embedding, sectioning, and the application of specific histological stains that highlight key anatomical features, including the epidermis, cortex, endodermis, vascular tissues, and root cap. The protocol also covers imaging techniques using confocal microscopy to obtain clear, reproducible images suitable for quantitative and qualitative analyses. This standardized approach facilitates comparative studies of root anatomy and development, offering a valuable tool for plant developmental biology research while ensuring the preservation of tissue morphology.
Methods in molecular biology (Clifton, N.J.)Abhinav Saurabh, Laurel G Mendelsohn, Tiffany M Powell-Wiley, Yvonne Baumer
Natural killer (NK) cells and monocytes are central components of the innate immune system, playing critical roles in host defense, immune surveillance, and inflammation. Functional alterations in these cell populations are implicated in cancer, infectious diseases, cardiovascular disease, and responses to environmental and psychosocial stressors. Accurate measurement of NK cell and monocyte distribution and function is therefore essential for both basic and translational immunology. In large-scale or longitudinal studies, cryopreserved peripheral blood mononuclear cells (PBMCs) are commonly used to enable standardized analyses across time and sites; however, cryopreservation and thawing can adversely affect cell viability and function. This chapter presents a reproducible protocol for the cryopreservation, thawing, and functional assessment of NK cells and monocytes from human PBMCs. PBMCs are cryopreserved using a 9:1 ratio of fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO), allowing long-term storage at ultra-low temperatures while preserving cellular integrity. The thawing process utilizes a nuclease to minimize cell clumping, enhancing cell recovery and viability. Following thawing, PBMCs are prepared for downstream functional assays. NK cell function is assessed using a standard degranulation assay following coculture with K562 target cells, providing a robust measure of cytotoxic capacity. Together, this protocol offers a practical guide for the reliable measurement of NK cell and monocyte distribution and function from cryopreserved PBMCs, supporting high-quality immunological analyses in clinical and population-based research.
Single-cell transcriptomics has revolutionized our understanding of cellular heterogeneity by enabling high-resolution gene expression profiling at the individual cell level. However, traditional single-cell RNA sequencing (scRNA-seq) lacks direct protein quantification, limiting comprehensive immunophenotyping. Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) overcomes this limitation by integrating antibody-derived tag (ADT) quantification with scRNA-seq, allowing simultaneous measurement of surface protein and gene expression from the same cell. This multimodal approach enhances immune cell characterization, revealing new functional states and rare subpopulations in complex biological systems. Here, we provide a detailed protocol for performing CITE-seq, from sample preparation to sequencing and data analysis. We highlight key experimental considerations, discuss challenges related to antibody selection and batch effects, and provide troubleshooting strategies to ensure robust and reproducible results. The integration of transcriptomic and proteomic data through CITE-seq provides unparalleled insights into cellular function, with broad applications in immunology, oncology, and systems biology.
Studying the transcriptome and the proteome of cells is essential for gaining a detailed understanding of cellular behavior, development, drug action, and disease progression. Spatial biology emerges to advance our ability to study the expression of molecules within tissues while preserving their natural spatial context. These cutting-edge technologies enable the mapping of thousands of individual cells in their original environment by detecting the location and biological quantity of cellular contents, such as RNAs and proteins. Here, we present a protocol to combine spatial transcriptomics (Xenium) and spatial proteomics (PhenoCycler-Fusion) within 8 days on the same tissue section to successfully study the expression of hundreds of RNAs and tens of proteins simultaneously. The combination of these two technologies and consequent integration of the two data layers together with high-resolution H&E images allows for the extraction of a maximum of information from a single tissue section. Application of this protocol and the resulting integrated data will help researchers to understand complex biological processes and disease mechanisms, supporting more nuanced research in molecular biology and pathology.
Methods in molecular biology (Clifton, N.J.)Pallavi Kanwar Galera, Raul Braylan
Immunophenotyping by multiparameter flow cytometry is a rapid and efficient technique to simultaneously assess and correlate multiple individual cell properties like size and internal complexity along with antigen expression in a population of cells. This method is utilized for rapid characterization of the blasts and classification of acute myeloid leukemia (AML) in both the peripheral blood (PB) and bone marrow (BM). This technique is not only useful in the initial diagnosis but also in monitoring and determining the prognosis of the disease through minimal residual disease (MRD) testing. This chapter provides an overview of procedures for specimen processing, staining, and immunophenotyping of AML and describes the principles of data analysis for AML classification and MRD testing.
Methods in molecular biology (Clifton, N.J.)Christopher A Lazarski, Anushree A Datar, Patrick J Hanley, Sandeep K Srivastava
Flow cytometric analysis can provide valuable information on the viability, phenotype, and therapeutic potential of cell therapy products using a minimal amount of cell numbers. In this chapter, we describe methods for the analysis of clinical products that incorporate extra considerations within the protocols to help maintain consistency and confidence of staining. This can include methods for evaluating the transduction efficiency for T cells expressing chimeric antigen receptors (CAR-T), characterizing the frequency of diverse lymphocyte populations within polyclonal cell cultures and postinfusion research samples, and measuring the activity or exhaustion in response to antigen-specific stimulation. This chapter also includes recommendations for storage and management of staining materials, frequency of compensation analysis, and reference standards.