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Combining Microwave Ablation With CAR-T-Cell Therapy in Tumor-Bearing Mouse Models
Microwave ablation (MWA) is a thermal ablation technique widely used for local tumor control that has the added potential to stimulate systemic anti-tumor immunity. Although MWA alone rarely eliminates recurrent or metastatic disease, its ability to remodel the tumor microenvironment makes it a promising partner for adoptive cell therapies such as chimeric antigen receptor (CAR)-T cells. However, reproducible protocols for combining these approaches remain limited. This protocol describes the integration of MWA with CAR-T therapy in tumor-bearing mouse models. Human hepatocellular carcinoma cell lines (Hep3B and SK-HEP-1) are inoculated subcutaneously into NOG mice to establish tumors. Localized MWA is performed at adjustable power and duration to induce partial or complete ablation. At defined intervals following MWA, CAR-T cells derived from healthy donor T cells and transduced with a lentiviral vector are injected intravenously. This experimental design uniquely separates MWA and CAR-T delivery, enabling precise evaluation of thermal preconditioning effects on the tumor microenvironment and subsequent CAR-T activity. By combining localized ablation with adoptive immunotherapy, the protocol provides a translationally relevant platform to optimize treatment timing, enhance CAR-T efficacy in solid tumors, and address key barriers in tumor immunology and cancer therapy.
Cycloheximide (CHX) Chase Assay to Examine Protein Half-life
Cycloheximide (CHX) is a small molecule derived from Streptomyces griseus that acts as fungicide. As a ribosome inhibitor, CHX can restrict the translation elongation of eukaryotic protein synthesis. Once protein synthesis is inhibited by CHX, the level of intracellular proteins decreases by degradation through the proteasome or lysosome system. Thus, the CHX chase assay is widely recognized and used to observe intracellular protein degradation and to determine the half-life of a given protein in eukaryotes. Here, we present a complete experimental procedure of the CHX chase assay.Graphical overview
Isolation and Co-culture of Paneth Cells and Intestinal Stem Cells
Crypts at the base of intestinal villi contain intestinal stem cells (ISCs) and Paneth cells, the latter of which work as niche cells for ISCs. When isolated and cultured in the presence of specific growth factors, crypts give rise to self-renewing 3D structures called organoids that are highly similar to the crypt-villus structure of the small intestine. However, the organoid culture from whole crypts does not allow investigators to determine the contribution of their individual components, namely ISCs and Paneth cells, to organoid formation efficiency. Here, we describe the method to isolate Paneth cells and ISCs by flow cytometry and co-culture them to form organoids. This approach allows the determination of the contribution of Paneth cells or ISCs to organoid formation and provides a novel tool to analyze the function of Paneth cells, the main component of the intestinal stem cell niche.
A Simple, Reproducible Procedure for Chemiluminescent Western Blot Quantification
Western blotting is a universally used technique to identify specific proteins from a heterogeneous and complex mixture. However, there is no clear and common procedure to quantify the results obtained, resulting in variations due to the different software and protocols used in each laboratory. Here, we have developed a procedure based on the increase in chemiluminescent signal to obtain a representative value for each band to be quantified. Images were processed with ImageJ and subsequently compared using R software. The result is a linear regression model in which we use the slope of the signal increase within the combined linear range of detection to compare between samples. This approach allows to quantify and compare protein levels from different conditions in a simple and reproducible way. Graphical overview
High-Dimensional Phospho-CyTOF Characterization of T-Cell Activation Responses in Whole Blood
Recent advances in single-cell technologies have provided limited insight into the role of protein phosphorylation in T-cell fate and function. Dysregulated protein phosphorylation is associated with adverse clinical outcomes, emphasizing the need for reliable methods to unravel the complexities of T-cell signal transduction and disease-related alterations. While flow cytometry is widely used, it is constrained by spectral overlap, limiting the number of protein targets for simultaneous analysis. To overcome this, we present a robust protocol for whole blood T-cell stimulation and subsequent analysis using mass cytometry by time-of-flight (CyTOF). CyTOF minimizes spillover into adjacent channels by employing highly pure, stable, heavy metal–conjugated antibodies for protein detection. This protocol offers a high-dimensional approach for phenotypic and phospho-protein characterization of key signaling pathways, including JAK/STAT, MAPK, PI3K/mTOR, PKC, and NF-κB. A key feature is the T-cell stimulation reagent, which mimics endogenous activation by engaging the T-cell receptor (TCR)/CD3 complex and providing co-stimulation via an anti-CD28 antibody. Further, we enhance reproducibility and enable batch processing through the implementation of the Prot1/Thaw-Lyse system for immediate cryopreservation of stimulated blood samples. By employing CyTOF, this method permits the simultaneous analysis of 31 protein targets with single-cell resolution, minimizing spillover and providing superior specificity, sensitivity, and resolution over flow cytometric methods. This approach facilitates the robust assessment of TCR activation and its effect on bystander populations, which has been challenging with spectral flow cytometry due to the limited availability of methanol-resistant fluorophores. This protocol is a precise and reproducible method for elucidating the downstream effects of T-cell stimulation and immune status, with significant potential for clinical applications, including the assessment of T-cell-targeted therapies.
Generation of Human Induced Pluripotent Stem Cell (hiPSC)-Derived Astrocytes for Amyotrophic Lateral Sclerosis and Other Neurodegenerative Disease Studies
Astrocytes are increasingly recognized for their important role in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS). In ALS, astrocytes shift from their primary function of providing neuronal homeostatic support towards a reactive and toxic role, which overall contributes to neuronal toxicity and cell death. Currently, our knowledge on these processes is incomplete, and time-efficient and reproducible model systems in a human context are therefore required to understand and therapeutically modulate the toxic astrocytic response for future treatment options. Here, we present an efficient and straightforward protocol to generate human induced pluripotent stem cell (hiPSC)-derived astrocytes implementing a differentiation scheme based on small molecules. Through an initial 25 days, hiPSCs are differentiated into astrocytes, which are matured for 4+ weeks. The hiPSC-derived astrocytes can be cryopreserved at every passage during differentiation and maturation. This provides convenient pauses in the protocol as well as cell banking opportunities, thereby limiting the need to continuously start from hiPSCs. The protocol has already proven valuable in ALS research but can be adapted to any desired research field where astrocytes are of interest.Key features• This protocol requires preexisting experience in hiPSC culturing for a successful outcome.• The protocol relies on a small molecule differentiation scheme and an easy-to-follow methodology, which can be paused at several time points.• The protocol generates >50 × 106 astrocytes per differentiation, which can be cryopreserved at every passage, ensuring a large-scale experimental output.Graphical overview
Ultrafast Isolation of Synaptic Terminals From Rat Brain for Cryo-Electron Tomography Analysis
Understanding the nanoscale organization and molecular rearrangement of synaptic components is critical for elucidating the mechanisms of synaptic transmission and plasticity. Traditional synaptosome isolation protocols involve multiple centrifugation and resuspension steps, which may cause structural damage or alter the synaptosomal fraction, compromising their suitability for cryo-electron tomography (cryo-ET). Here, we present an ultrafast isolation method optimized for cryo-ET that yields two types of synaptosomal fractions: synaptosomes and synaptoneurosomes. This streamlined protocol preserves intact postsynaptic membranes apposed to presynaptic active zones and produces thin, high-quality samples suitable for in situ structural studies. The entire procedure, from tissue homogenization to vitrification, takes less than 15 min, offering a significant advantage for high-resolution cryo-ET analysis of synaptic architecture.
Cochlear Organ Dissection, Immunostaining, and Confocal Imaging in Mice
The organ of Corti, located in the inner ear, is the primary organ responsible for animal hearing. Each hair cell has a V-shaped or U-shaped hair bundle composed of actin-filled stereocilia and a kinocilium supported by true transport microtubules. Damage to these structures due to noise exposure, drug toxicity, aging, or environmental factors can lead to hearing loss and other disorders. The challenge when examining auditory organs is their location within the bony labyrinth and their small and fragile nature. This protocol describes the dissection procedure for the cochlear organ, followed by confocal imaging of immunostained endogenous and fluorescent proteins. This approach can be used to understand hair cell physiology and the molecular mechanisms required for normal hearing.
Generation of Intestinal Epithelial Monolayers From Single-Cell Dissociated Organoids
Intestinal organoids are generated from intestinal epithelial stem cells, forming 3D mini-guts that are often used as an in vitro model to evaluate and manipulate the regenerative capacities of intestinal epithelial stem cells. Plating 3D organoids on different substrates transforms organoids into 2D monolayers, which self-organize to form crypt-like regions (which contain stem cells and transit amplifying cells) and villus-like regions (which contain differentiated cells). This “open lumen” organization facilitates multiple biochemical and biomechanical studies that are otherwise complex in 3D organoids, such as drug applications to the cell’s apical side or precise control over substrate protein composition or substrate stiffness. Here, we describe a protocol to generate homogenous intestinal monolayers from single-cell intestinal organoid suspension, resulting in de novo crypt formation. Our protocol results in higher viability of intestinal cells, allowing successful monolayer formation.
An Optimized Protocol for Simultaneous Propagation of Patient-derived Organoids and Matching CAFs
Recurrent hormone receptor-positive (HR+) breast cancer is a leading cause of cancer mortality in women. Recurrence and resistance to targeted therapies have been difficult to study due to the long clinical course of the disease, the complex nature of resistance, and the lack of clinically relevant model systems. Existing models are limited to a few HR+ cell lines, organoid models, and patient-derived xenograft models, all lacking components of the human tumor microenvironment. Furthermore, the low take rate and loss of estrogen receptor (ER) expression in patient-derived organoids (PDOs) has been challenging. Our protocol allows simultaneous isolation of PDOs and matching cancer-associated fibroblasts (CAFs) from primary and metastatic HR+ breast cancers. Importantly, our protocol has a higher take rate and enables long-term culturing of PDOs that retain ER expression. Our matching PDOs and CAFs will provide researchers with a new resource to study the influence of the tumor microenvironment on various aspects of cancer biology such as cell growth and drug resistance in HR+ breast cancer.
Preparation and Characterization of Lipid Nanoparticles Co-loaded With DNA and Nitro-Oleic Acid
Lipid nanoparticles (LNPs) are powerful carriers for nucleic acid delivery, but plasmid DNA-loaded LNPs (pDNA-LNPs) have been limited by inflammation and toxicity. We showed that standard pDNA-LNPs activate the cGAS–STING pathway, leading to severe immune responses and mortality in mice. To overcome this, we co-loaded nitro-oleic acid (NOA), an endogenous STING inhibitor, into pDNA-LNPs. NOA-pDNA-LNPs mitigated inflammation, enabled safe in vivo delivery, and supported sustained gene expression for months. Here, we present a detailed protocol for producing and characterizing NOA-pDNA-LNPs to facilitate safer, long-term gene delivery applications.
Cloning a Chloroplast Genome in Saccharomyces cerevisiae and Escherichia coli
Chloroplast genomes present an alternative strategy for large-scale engineering of photosynthetic eukaryotes. Prior to our work, the chloroplast genomes of Chlamydomonas reinhardtii (204 kb) and Zea mays (140 kb) had been cloned using bacterial and yeast artificial chromosome (BAC/YAC) libraries, respectively. These methods lack design flexibility as they are reliant upon the random capture of genomic fragments during BAC/YAC library creation; additionally, both demonstrated a low efficiency (≤ 10%) for correct assembly of the genome in yeast. With this in mind, we sought to create a highly flexible and efficient approach for assembling the 117 kb chloroplast genome of Phaeodactylum tricornutum, a photosynthetic marine diatom. Our original article demonstrated a PCR-based approach for cloning the P. tricornutum chloroplast genome that had 90%–100% efficiency when screening as few as 10 yeast colonies following assembly. In this article, we will discuss this approach in greater depth as we believe this technique could be extrapolated to other species, particularly those with a similar chloroplast genome size and architecture.
Rapid Isolation and Flow Cytometry Analysis of Murine Intestinal Immune Cells After Chemically Induced Colitis
Chemically induced murine colitis models are widely used to understand intestinal homeostasis and inflammatory responses during acute and chronic gut inflammation, such as inflammatory bowel disease (IBD). Resident populations of immune cells, together with those recruited during an inflammatory response, maintain intestinal immunity by mounting an effective immune response to enteropathogenic microbes while at the same time maintaining tolerance against commensals. To better understand the disease mechanism, studying different immune cell populations and their dynamic changes during infection and inflammation is essential. However, isolating healthy and viable immune populations, particularly hyperactivated neutrophils and macrophages from the inflamed gut (i.e., active disease site), is challenging as tissues are usually subjected to rigorous enzymatic digestion for an extended period. Here, we describe a method that uses a cell dissociator (Medimachine II from Syntec International) to separate intestinal tissue after short enzymatic digestion to obtain a single-cell suspension. This technique facilitates the isolation of immune cells from mouse intestinal tissues in high quantity and with superior viability in a very short time frame. This protocol delivers 80%–90% cell viability, which is 1.5 to 2-fold higher than conventional methods of isolating cells from inflamed mouse colons. The composition, phenotype, activation state, and gene expression profile of cells isolated using this protocol can be assessed by using multiple methods, including, but not limited to, flow cytometry, quantitative PCR, immunoblotting, mass spectrometry, single-cell RNA sequencing, and functional readouts such as reactive oxygen species (ROS) production.
A Protocol for Weighted Gene Co-expression Network Analysis With Module Preservation and Functional Enrichment Analysis for Tumor and Normal Transcriptomic Data
Weighted gene co-expression network analysis (WGCNA) is widely used in transcriptomic studies to identify groups of highly correlated genes, aiding in the understanding of disease mechanisms. Although numerous protocols exist for constructing WGCNA networks from gene expression data, many focus on single datasets and do not address how to compare module stability across conditions. Here, we present a protocol for constructing and comparing WGCNA modules in paired tumor and normal datasets, enabling the identification of modules involved in both core biological processes and those specifically related to cancer pathogenesis. By incorporating module preservation analysis, this approach allows researchers to gain deeper insights into the molecular underpinnings of oral cancer, as well as other diseases. Overall, this protocol provides a framework for module preservation analysis in paired datasets, enabling researchers to identify which gene co-expression modules are conserved or disrupted between conditions, thereby advancing our understanding of disease-specific vs. universal biological processes.
Generation of 3D Human iPSC-Derived Multi-Cell Type Neurospheres for Studying Neuron, Astrocyte, and Microglia Crosstalk
Three-dimensional (3D) human brain tissue models derived from induced pluripotent stem cells (iPSCs) have transformed the study of neural development and disease in vitro. While cerebral organoids offer high structural complexity, their large size often leads to necrotic core formation, limiting reproducibility and challenging the integration of microglia. Here, we present a detailed, reproducible protocol for generating multi-cell type 3D neurospheres that incorporate neurons, astrocytes, and optionally microglia, all derived from the same iPSCs. While neurons and astrocytes differentiate spontaneously from neural precursor cells, generated by dual SMAD-inhibition (blocking BMP and TGF-b signaling), microglia are generated in parallel and can infiltrate the mature neurosphere tissue after plating neurospheres into 48-well plates. The system supports a range of downstream applications, including functional confocal live imaging of GCaMP6f after adeno-associated virus (AAV) transduction of neurospheres or immunofluorescence staining after fixation. Our approach has been successfully implemented across multiple laboratories, demonstrating its robustness and translational potential for studying neuron–glia interactions and modeling neurodegenerative processes.
Click-qPCR: A Simple Tool for Interactive qPCR Data Analysis
Real-time quantitative PCR (qPCR) is a pivotal technique for analyzing gene expression and DNA copy number variations. However, the limited availability of user-friendly software tools for qPCR data analysis presents a significant challenge for experimental biologists with limited computational skills. To address this issue, we developed Click-qPCR, a user-friendly and web-based Shiny application for qPCR data analysis. Click-qPCR streamlines ΔCq and ΔΔCq calculations using user-uploaded CSV data files. The interactive interface of the application allows users to select genes and experimental groups and perform Welch’s t tests and one-way analysis of variance with Dunnett’s post-hoc test for pairwise and multi-group comparisons, respectively. Results are visualized via interactive bar plots (mean ± standard deviation with individual data points) and can be downloaded as publication-quality images, along with summary statistics. Click-qPCR empowers researchers to efficiently process, interpret, and visualize qPCR data regardless of their programming experience, thereby facilitating routine analysis tasks. Click-qPCR Shiny application is available at https://kubo-azu.shinyapps.io/Click-qPCR/, while its source code and user guide are available at https://github.com/kubo-azu/Click-qPCR.
Isolation and Imaging of Microvessels From Brain Tissue
Proper brain function depends on the integrity of the blood–brain barrier (BBB), which is formed by a specialized network of microvessels in the brain. Reliable isolation of these microvessels is crucial for studying BBB composition and function in both health and disease. Here, we describe a protocol for the mechanical dissociation and density-based separation of microvessels from fresh or frozen human and murine brain tissue. The isolated microvessels retain their molecular integrity and are compatible with downstream applications, including fluorescence imaging and biochemical analyses. This method enables direct comparisons across species and disease states using the same workflow, facilitating translational research on BBB biology.
Use of a High-Affinity Ubiquitin-Binding Domain to Detect and Purify Ubiquitinated Substrates and Their Interacting Proteins
OtUBD is a high-affinity ubiquitin-binding domain (UBD) derived from a large protein produced by the microorganism Orientia tsutsugamushi. The following protocol describes a step-by-step process for the enrichment of ubiquitinated proteins from baker's yeast and mammalian cell lysates using OtUBD. The OtUBD affinity resin can strongly enrich both mono- and poly-ubiquitinated proteins from crude lysates. The protocol further describes the use of different buffer formulations to specifically enrich for proteins covalently modified by ubiquitin with or without proteins that associate with them. Combining different OtUBD-mediated enrichment protocols with liquid chromatography–tandem mass spectrometry (LC–MS/MS) helps distinguish the pool of covalently ubiquitinated proteins (the ubiquitinome) from ubiquitin- or ubiquitinated protein-interacting proteins (the ubiquitin interactome). The OtUBD tool described in the protocol has been used successfully with downstream applications such as immunoblotting and differential proteomics. It provides researchers with a versatile and economical tool for the study of ubiquitin biology.
Verification of N-Linked Glycosylation of Proteins Isolated from Plant or Mammalian Cell Lines Using PNGase Enzyme
N-glycosylation is a ubiquitous post-translational modification (PTM) that regulates protein folding, stability, and biological function. Accurate identification and validation of N-glycosylation are therefore critical for understanding how glycosylation modulates protein activity. Here, we present a robust workflow for analyzing protein N-glycosylation in both animal and plant systems using peptide-N4-(N-acetyl-β-glucosaminyl) asparagine-amidase A and F (PNGase A and PNGase F). After enzymatic cleavage of the asparagine-linked N-glycans, samples are analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting (WB) to detect shifts in apparent molecular weight (MW) indicative of deglycosylation. Key steps include denaturing the protein to expose glycosylation sites, optimizing buffer conditions for PNGase F and A treatment, and comparing glycosylated vs. deglycosylated forms by electrophoretic mobility. A troubleshooting guide addresses common challenges, including incomplete deglycosylation and low transfer efficiency during WB, offering practical solutions to ensure reliable results. This protocol provides researchers with a standardized, cost-effective framework for investigating protein N-glycosylation in diverse systems, from cell lysates to purified proteins, in both animal and plant models.
Preparation of Chromatin Fragments From Human Cells for Cryo-EM Analysis
Eukaryotic genomic DNA is packaged into chromatin, which plays a critical role in regulating gene expression by dynamically modulating its higher-order structure. While in vitro reconstitution approaches have offered valuable insights into chromatin organization, they often fail to fully capture the native structural context found within cells. To overcome this limitation, we present a protocol for isolating native chromatin fragments from human cells for cryo-electron microscopy (cryo-EM) analysis. In this method, chromatin from formaldehyde-crosslinked human HeLa S3 nuclei is digested with micrococcal nuclease (MNase) to generate mono- and poly-nucleosome fragments. These fragments are subsequently fractionated by sucrose-gradient ultracentrifugation and prepared for cryo-EM. The resulting chromatin fragments retain native-like nucleosome–nucleosome interactions, facilitating structural analyses of chromatin organization under near-physiological conditions.
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