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Cell engineering Cell imaging Cell isolation and culture Cell metabolism Cell movement Cell signaling Cell staining Cell structure Cell Transplantation Cell viability Cell-based analysis Model organism culture Organelle isolation Single cell analysis Tissue analysis
A Python-Based Workflow for Image-Based Single-Cell Phenotypic Profiling From Fluorescence Microscopy Images
While advances in omics technologies have greatly improved our understanding of cellular heterogeneity, there is an increasing need for complementary approaches that capture the spatial organization and structural dynamics of cells. Image-based single-cell phenotypic profiling provides quantitative information on cell morphology and organelle organization, offering valuable insights into cellular function and regulation. Although numerous image analysis tools are available, establishing a complete analysis workflow, from image preprocessing and segmentation to feature extraction and multivariate analysis, often requires substantial computational expertise and software integration. Here, we describe a Python-based workflow for image-based single-cell phenotypic profiling from immunofluorescence microscopy images and provide a detailed protocol for its implementation. Using synchronized HeLa cells with drug-induced mitotic spindle defects as an example, the workflow covers image loading, cell segmentation, quantitative feature extraction, profile integration, dimensionality reduction, clustering, and data visualization. The protocol is accompanied by example datasets, annotated Jupyter Notebooks, and instructions for execution in either a local Python environment or Google Colab, facilitating straightforward implementation and customization. By integrating the entire analysis pipeline within a single coding environment, this workflow enables reproducible and accessible single-cell morphological profiling without requiring specialized imaging equipment or extensive programming expertise. The workflow therefore provides a practical platform for studying cell morphology, organelle organization, and cellular dynamics across a broad range of biological applications.
Quantification of Asymmetric Division in Cancer Cells via a Flow Cytometry and Cell Sorting–Based Protocol
Asymmetric partitioning of cellular components during division contributes to generating phenotypic heterogeneity and plasticity in genetically identical cells, like bacteria or cancer cells. Traditionally, this partitioning noise has been investigated using live-cell time-lapse confocal microscopy; however, while imaging offers high spatial resolution, it suffers from low statistical throughput and labor-intensive cell tracking. To overcome these limitations, we present a protocol that combines high-throughput flow cytometry and sorting with a theoretical model to precisely quantify partitioning noise during cell division. This method details the experimental setup for measuring cellular components in dividing populations, the data acquisition pipeline, and the implementation of the mathematical framework to extract inheritance parameters. Applicable to both suspension and adherent cells, we have validated our approach against live-cell imaging of adherent colon cancer cells. Hence, this flow cytometry–based approach offers a highly scalable, accessible, and statistically robust alternative to traditional imaging. It is widely applicable across diverse cell lines and biological systems, significantly advancing our capacity to study partitioning-driven phenotypic heterogeneity.
Establishment and Cryopreservation of Primary Fibroblast Cultures From Livestock Ear-Tissue Explants
Primary fibroblast cell culture is widely used in livestock research as a renewable genetic source for applications such as cell banking, molecular analyses, and functional studies [1]. Establishing high-quality primary culture requires careful tissue collection, aseptic processing, and optimized cell culture conditions to maximize growth and minimize contamination [2]. This protocol describes a standardized method for establishing primary fibroblast cultures from livestock species, representing multiple breeds, including cattle, swine, sheep, and goats. Although the ear is the primary tissue source described due to its easy accessibility and minimal invasiveness, this protocol may also be used with similar skin or tissue types if needed. Procedures for tissue collection, sample preparation, explant culture, routine maintenance, passaging, and cryopreservation are detailed to support the generation and proliferation of healthy fibroblast cultures suitable for downstream research applications.
Suspension-Based Intracellular Immunofluorescence Staining Coupled With Genome-Wide CRISPR-Cas9 Screening to Identify Regulators of O-GlcNAcylation
Protein O-GlcNAcylation is a dynamic and reversible post-translational modification that regulates diverse cellular processes, including transcription, signal transduction, metabolism, and cell fate determination. Systematic identification of genes that modulate global O-GlcNAc levels remains technically challenging at the genome scale. Here, we describe a pooled CRISPR-Cas9 screening protocol that combines the human genome-scale CRISPR knockout (GeCKO) v2 knockout library with intracellular immunofluorescence staining using the anti-O-GlcNAc antibody RL2 and fluorescence-activated cell sorting (FACS). In this workflow, HEK293T cells are transduced with the GeCKO v2 lentiviral library at a low multiplicity of infection to ensure predominantly single-sgRNA integration. Following puromycin selection, cells are fixed, permeabilized, and stained in suspension with RL2. The top 5% of cells with the highest intracellular RL2 fluorescence are collected as the RL2-high population, while a corresponding unsorted/input sample is retained as the reference for downstream sgRNA enrichment analysis. Genomic DNA is recovered from the RL2-high and unsorted/input samples, and integrated sgRNA cassettes are amplified through a two-step PCR and library-preparation workflow for Illumina sequencing. This protocol enables the identification of candidate genes whose knockout is associated with increased intracellular RL2 fluorescence.
Mouse Intratongue Injection With Cancer Cells—An Orthotopic Model for Head and Neck Squamous Cell Carcinoma
Syngeneic and orthotopic injection of cancer cells in mice is a gold standard model in pre-clinical cancer research. Herein, we detail a protocol of orthotopic intratongue injection as a model for upper respiratory tract cancer studies. This model recapitulates tumor growth and invasion in an orthotopic site, the oral cavity mucosa. It can be experimentally designed in a syngeneic context or used to study patient-derived cells in immunocompromised animals. Procedurally, mice are anesthetized and injected with a small volume of cancer cell suspension near the lateral border of the tongue, right under the mucous membrane. Post-injection, animal welfare is carefully attended to by appropriate analgesia, while providing soft food for the duration of the experiment to ensure the ability of the mice to eat ad libitum. Tongues and potential metastatic organs are dissected and can be sampled and analyzed using relevant state-of-the-art methods, including -omics and imaging approaches. We describe a broad range of distinct tumor profiling and analysis methods applicable to this model. These include physical measurements of elasticity with atomic force microscopy on fresh tissue, hematoxylin-eosin staining and standard immunohistochemistry, and confocal microscopy applied to paraffin-embedded tongue sections. This model is orthotopic, accessible without surgery, easy to monitor, and rapid to perform.
Anther Dissection and Confocal Imaging of Developing and Mature Pollen in Arabidopsis
Pollen development is a fundamental process underlying plant fertility that involves a series of coordinated cellular transitions from microspore mother cells (MMCs) to mature pollen grains. Conventional approaches for studying this process often require tissue sectioning, extensive sample preparation, and specialized instrumentation operated by trained personnel, making them labor-intensive, time-consuming, costly, and poorly suited for high-throughput analysis. Here, we describe simpler protocols for preparing and imaging both developing pollen lineage cells and mature pollen grains from Arabidopsis. By combining targeted anther dissection with gentle extrusion of pollen cells from anthers, fluorescent staining, and confocal microscopy, our protocol makes pollen cells at different developmental stages accessible for high-resolution imaging, enabling visualization of cellular morphology and protein localization, as well as fluorescence-based protein quantification. We also describe a complementary workflow for imaging mature pollen grains that provides a rapid alternative to scanning electron microscopy (SEM) for examining pollen surface structures and patterns. Together, these protocols offer practical approaches for investigating the cellular and molecular processes underlying pollen development and for characterizing the surface morphology of mature pollen.
Transcriptomic Signature-Guided EGFP Labeling of Alveolar Epithelial Cells via RNA Sensing in Mouse Lungs In Vivo and Human Idiopathic Pulmonary Fibrosis Lung Slices Ex Vivo
Single-cell RNA sequencing has revealed transient and disease-associated alveolar epithelial states that are difficult to access with conventional transgenic approaches. Here, we describe a programmable RNA-sensing workflow based on CellREADR [cell access through RNA sensing by endogenous adenosine deaminases acting on RNA (ADAR)] to couple a selected cellular RNA signature to enhanced green fluorescent protein (EGFP) translation. The protocol covers sequence-encoded sensing RNA (sesRNA) design and cloning, in vitro validation of sesRNA-dependent EGFP expression in HEK293T cells, production and heparin-column purification of adeno-associated virus variant 6.2FF (AAV6.2FF), delivery to mouse lungs, and application to human idiopathic pulmonary fibrosis (IPF) precision-cut lung slices (PCLS). Sensors targeting Sftpc, Pdpn, or Sprr1a can be used to label alveolar type 2, alveolar type 1, and injury-induced Krt8+ alveolar differentiation intermediate cells in mice and KRT17+KRT5- aberrant basaloid cells in humans, respectively. Because cell specificity is programmed by the sensor sequence rather than a cell type–specific promoter or newly generated mouse line, this approach can be rapidly adapted to emerging transcriptomic targets in both experimental mouse models and human lung tissues, enabling cross-species validation and translational investigation of disease-associated epithelial states.
Myoclone Assay: Clonogenic Expansion of Activating Muscle Stem Cells
Muscle regeneration depends on the function of adult muscle stem cells (MuSCs). During homeostasis, MuSCs reside in a quiescent state of prolonged reversible cell cycle exit. Upon muscle injury, MuSCs exit the quiescent state and activate to enter the cell cycle. Subsequently, they undergo rounds of clonogenic expansion before differentiating to form new muscle fibers. Any impairment to the MuSCs’ ability to switch between these cell states can impair muscle regeneration. To study MuSC function, it is therefore important to quantitatively assess MuSCs’ capacity to switch between cell states. Numerous robust methods have been developed that assess the activation and proliferation capacity of MuSCs. However, these methods often require thousands of cells, and MuSCs are rare. Here, we present a protocol to assess MuSC activation using only 60 cells per sample by monitoring the parallel clonogenic expansion of single MuSCs. Single primary MuSCs are seeded into 96-well half-area microplates at a 1-cell-per-well density, which takes as little as 2 min per sample. The cells are then incubated for 8 days in the presence of growth factors, stained with Hoechst, and imaged by inverse fluorescence microscopy. We demonstrate that relative clonogenic expansion data provide results that are similar to published data obtained using other methods, such as cumulative EdU incorporation assays. This myoclone protocol enables the quantitative assessment of MuSC activation using only 60 cells, in a time-efficient and cost-effective manner, and thereby can provide new insights into MuSC function.
Monitoring Protein Secretion in Living Cells With a HiBiT-Integrated RUSH System
The cellular secretory pathway is a fundamental physiological process; however, precise, efficient, and quantitative methods for monitoring this process remain rarely explored. Although several classic approaches have been developed to investigate secretory trafficking, the RUSH (Retention Using Selective Hooks) system currently represents the most advanced technique. Here, we introduce a method termed the HiBiT-integrated RUSH system and provide a detailed protocol for its application. This approach enables real-time monitoring of the secretion of cell membrane receptors such as ACE2, PDL1, NTCP, and CD209. By converting the cellular secretion process into a quantitative curve, our method offers an objective means for result interpretation. The HiBiT-integrated RUSH system is particularly effective for studying the transport dynamics of various cell membrane receptors, thereby facilitating the identification of both common and specific regulators of the cellular secretory pathway.
Visualizing Cell Type–Specific Glycan Profiles in Tissue Sections With Glycan Painting
Glycans play critical roles in numerous biological processes. Therefore, visualizing the spatial distribution of glycans within cells and tissues is essential for understanding tissue architecture and cellular identity. Glycan painting is a novel tissue-staining method that employs three fluorescently labeled lectins with different glycan-binding specificities to visualize cell type–specific glycan profiles as unique color patterns. In this study, the protocol was validated using formalin-fixed paraffin-embedded sections of mouse embryos. Although Glycan painting is a qualitative method, each cell population that is difficult to distinguish with a single lectin can be differentiated as unique color patterns generated from the combined images of three fluorescent lectins. Moreover, because it does not require costly reagents or specialized equipment, Glycan painting provides a cost-effective and broadly accessible approach for full-color tissue imaging and cell-type discrimination.
Microtube-Assisted Paraffin Embedding and Sectioning of Fragile 3D Tumor Spheroids for High-Quality Histology
Three-dimensional (3D) spheroid models have become essential in cancer biology, drug screening, and tissue engineering. However, their small size, fragile structure, and tendency to disintegrate during routine histoprocessing present persistent technical challenges. Conventional paraffin embedding often results in tissue fragmentation, loss of spatial orientation, and poor section quality, whereas cryosectioning often compromises cellular morphology. Here, we present a robust, cost-effective protocol for preserving and sectioning fragile 3D spheroids, resulting in high-quality histological sections with intact architecture and excellent cellular detail. The method involves optimized handling and embedding procedures that stabilize spheroids during standard formalin fixation, paraffin infiltration, and microtomy, eliminating mechanical distortion and preserving spherical integrity for consistent sectioning. We demonstrate the successful application of this protocol across different tumor spheroids derived from distinct tissue types, including MDA-MB-231 (breast carcinoma), Caco-2 (colorectal adenocarcinoma), and A549 (lung carcinoma) cell lines, with subsequent compatibility with hematoxylin and eosin (H&E) staining protocols. This protocol is compatible with immunohistochemistry (IHC) and immunofluorescence (IF). In our qualitative comparison with conventional methods, we found that our approach effectively limits sample loss, promotes consistent inter-section reproducibility, and preserves fine structural features, such as necrotic cores, proliferative zones, and extracellular matrix components. This protocol provides a reliable, accessible solution for routine histological analysis of fragile 3D spheroids, facilitating more accurate morphological assessment in translational research settings.
A Dissociation-Based Seahorse XF Workflow for Quantifying Averaged Bioenergetics in Anchorage-Independent Cancer Cell Clusters
Cell clustering during anchorage-independent growth (AIG) is a key hallmark of malignant progression, supporting survival under detachment during metastatic dissemination. However, bioenergetic profiling of cell clusters grown under AIG conditions remains technically challenging, in part because of steep nutrient and oxygen gradients and irregular cell cluster shapes that complicate Seahorse XF assay normalization. Here, we present a dissociation-based Seahorse XF96/XF Pro method that enables high-throughput quantification of the average bioenergetic state of anchorage-independent cancer cell clusters. In this workflow, clusters generated in ultra-low adherent plates are enzymatically and mechanically dissociated into a single-cell suspension immediately prior to Seahorse XF Mito Stress Test analysis on poly-D-lysine-coated 2D microplates. This approach ensures that all viable cells throughout each cluster contribute equally to oxygen consumption rate (OCR) measurements and reduces the need for complex post hoc normalization. We further apply this workflow to an in vitro metastasis model in which A549 lung cancer cells are studied as attached, detached (anchorageindependent), and reattached populations, mimicking primary tumor cells, circulating/clustered cells, and secondary lesions, respectively. Detached cells exhibit reduced mitochondrial membrane potential and diminished basal and maximal OCR, whereas reattached cells largely restore membrane potential and respiratory capacity to levels comparable to attached cells, demonstrating that detachment-induced mitochondrial dysfunction is reversible upon reattachment. These findings align with previous work showing that anchorage-independent and clustered cancer cells undergo reversible metabolic reprogramming, including suppression of oxidative metabolism, engagement of reductive pathways to support redox homeostasis, and altered ROS handling to facilitate metastatic colonization. Collectively, this dissociation-based Seahorse workflow provides a practical and accessible platform to interrogate metabolic adaptations to anchorage-independent growth and cell clustering and identify bioenergetic vulnerabilities of metastasis-relevant cancer cell states.
Serial Cryosectioning for the Spatial Transcriptomics of Plant Tissues
Spatial transcriptomics enables genome-wide gene expression profiling while preserving tissue architecture, making it a powerful approach for studying plant developmental transitions. However, preparing small and structurally complex plant tissues for spatial transcriptomics remains technically challenging because samples must be rapidly preserved, precisely oriented, serially sectioned, and accurately positioned within the limited capture area of the Visium slides. Here, we describe an optimized workflow for cryo-embedding, serial cryosectioning, section placement, and data analysis of small plant samples for 10x Genomics Visium spatial transcriptomics. Using maize seedling shoot apices as the target, this protocol includes preparation of custom molds for optimal cutting temperature embedding, rapid fresh sample embedding, serial cryosectioning, section-position marking for Visium HD workflows, and morphological quality assessment of replicate tissue slides before transcript capture. The associated data analysis workflow includes Space Ranger processing, Seurat-based normalization and Harmony integration, anatomical domain annotation, pseudobulk and developmental trend analyses, RNA velocity, pseudotime analysis, transcription factor network analysis, single-cell reference mapping, and 3D transcriptome reconstruction. This computational workflow was developed and tested using maize Visium V1 data, but not Visium HD data. This protocol was used to generate serial spatial transcriptomes of maize shoot apices and developing leaf primordia, enabling reconstruction of gene expression transitions from the shoot apical meristem to sequential leaf developmental stages. The approach is also applicable to other small plant tissues, including Arabidopsis first true leaves and Marchantia thalli.
PIC-RNA-seq for Region-Specific Transcriptomic Analysis of Chicken Limb Bud Progenitors
Region-specific RNA sequencing is a powerful approach for investigating tissue differentiation and dynamic changes in gene expression during embryonic development. The chicken embryo has long served as an important model system in developmental biology. However, the limited availability of tissue-specific reporter lines makes region-specific RNA-seq approaches particularly valuable in this organism. Here, we applied photo-isolation chemistry-based RNA sequencing (PIC-RNA-seq) to the somatic lateral plate mesoderm (sLPM) before the emergence of limb bud progenitor cells (LPCs) and to early LPCs in chicken embryos. These analyses revealed the upregulation of multiple genes, including Hox genes, in LPCs, suggesting the initiation of their regional patterning program. This workflow enables visualization of dynamic changes in the gene expression profile of LPCs and should also be applicable to other tissues in avian embryos.
Disease Modeling in iPSC-Derived Human Bone Marrow Organoids
Human bone marrow organoids provide a tractable three-dimensional platform for modeling hematopoiesis and hematologic disease in a human niche–like context. Here, we describe a stepwise protocol for utilizing human induced pluripotent stem cell (iPSC)-derived bone marrow organoids that support autonomous hematopoiesis for hematopoietic disease modeling, mouse xenograft hematopoiesis, and drug sensitivity testing. The workflow combines embryoid body formation, early mesoderm/angiogenic induction under hypoxia, hemogenic endothelial commitment, maturation within a collagen-containing hydrogel, and subsequent suspension culture as individual organoids. The resulting organoids contain endothelial, stromal, and hematopoietic components and reproduce key structural and cellular features of human marrow. We further describe procedures for engraftment of normal donor- or patient-derived CD34+ cells and implantation of mature organoids under the renal capsule of immunodeficient mice to assess in vivo hematopoietic maintenance. In prior applications of this platform, donor-derived CD34+ cells were shown to engraft within the organoid niche and undergo multilineage differentiation, enabling detection of selective erythroid defects caused by DDX41 deficiency and assessment of therapeutic suppression of JAK2V617F-mutant patient-derived hematopoietic cells in a human marrow–like microenvironment. This protocol, therefore, enables disease modeling, in vivo xenograft assessment, and ex vivo functional analysis of patient-derived hematopoietic cells using relatively small input samples.
Quantitative Colocalization Analysis in Fluorescence Microscopy
Spatial organization of macromolecules is fundamental to cellular function, with colocalization providing key insights into molecular interactions and biological processes. However, quantification remains challenging due to diverse localization patterns and irregular sample geometries. Here, we present a protocol for analyzing colocalization between two fluorescent probes using coAnalyzer, a MATLAB-based software package. coAnalyzer features a user-friendly graphical interface and supports region of interest (ROI) selection, image merging, line scanning, signal isolation, scatterplot generation, and quantitative analysis. coAnalyzer enables colocalization analysis of any two fluorophores, regardless of the proteins or dyes involved. Its broad applicability across a wide range of organisms and sample types demonstrates the robustness, flexibility, and versatility of the platform.
Applications of OptoProfilin in Living Cells for the Imaging of Focal Adhesions and Stress-Associated Phenotypes
Cellular stress induces profound changes in cytoskeletal organization and biomolecular condensate formation. Traditional approaches for monitoring cellular stress often require multi-component biosensors, endpoint staining procedures, or indirect biochemical measurements. Here, we describe a protocol for the use of OptoProfilin, a genetically encoded single-component optogenetic biosensor derived from Profilin-1 fused to Cryptochrome 2 (Cry2) and mCherry. Following transient expression in mammalian cells, OptoProfilin exhibits light-dependent localization to focal adhesions under non-stressed conditions and transitions to punctate condensates under energetic, oxidative, osmotic, and senescence-associated stress conditions. The protocol includes transient transfection, induction of cellular stress, live-cell imaging, immunofluorescence validation, and quantitative image analysis. While this protocol describes imaging on a Leica widefield fluorescence microscope, it can readily be extended to other microscopy platforms. As a stand-alone biosensor that produces visually distinct responses in stressed versus non-stressed cells, OptoProfilin provides a convenient platform for investigating stress-associated cytoskeletal remodeling and biomolecular condensate formation.
Identifying Differentially Expressed Proteins via Plasma Exosomal Proteomics
Extracellular vesicle (EV) proteomics can be used to study intercellular communication and find biomarkers of non-invasive diseases. Traditional separation methods (ultracentrifugation, size exclusion chromatography) and data-dependent acquisition (DDA) mass spectrometry usually have the drawbacks of copurification of pollutants, poor reproducibility, and insufficient sampling of low-abundance peptides. This protocol describes a workflow for label-free extracellular vesicle proteomics, which combines size exclusion chromatography for vesicle separation, data-independent acquisition (DIA) for deep discovery, and parallel reaction monitoring (PRM) for targeted verification. Plasma-derived extracellular vesicles are treated by standardized lysis, digestion, and LC-MS/MS procedures, so that the protein group of extracellular vesicles can be identified and quantified robustly. The main advantages of this scheme are that it can achieve high quantitative reproducibility, unbiased detection of low-intensity peptides, and seamless transition from discovery to targeted verification, while also being suitable for liquid biopsy samples and various cardiovascular diseases.
3D Iterative Immunofluorescence Imaging on Whole-Mount Samples
Multicellular organization relies on reciprocal interactions between molecular events, such as gene expression and protein state, and higher-scale properties, such as spatial patterning and tissue architecture. Understanding these processes requires methods that enable quantitative measurements at subcellular resolution, while maintaining the three-dimensional tissue organization. Conventional immunofluorescence imaging captures spatial information but is limited to the number of fluorescence markers that can be imaged simultaneously, whereas dissociation-based single-cell approaches can profile multimodal cellular states but lack positional information. Here, we describe 3D in toto iterative immunofluorescence imaging, termed 3D-4i, which enables up to ten-plex protein and protein state measurements in early zebrafish embryos. Leveraging sample immobilization on 96-well plates together with a gentle liquid handling system and high-content spinning disc confocal microscopy, this method comprises repeated rounds of antibody staining, optical clearing, confocal imaging, and antibody elution. Subsequent image analysis allows segmentation of nuclei and cells, extraction of quantitative single-cell features, and integration of molecular measurements with spatial context. Altogether, 3D-4i provides a scalable platform for investigating diverse biological processes in intact embryos, while maintaining both subcellular resolution and three-dimensional context.
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