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Current Issue in 2025
Volume: 15, Issue: 20
Cancer Biology
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.
Cell Biology
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.
Studying Cargo Transport Using RudLOV
Most membrane and secreted proteins are transported from the endoplasmic reticulum (ER) to the Golgi apparatus and subsequently directed to their final destinations in the cell. However, the mechanisms underlying transport and cargo sorting remain unclear. Recent advancements in optical microscopy, combined with synchronized cargo protein release methods, have enabled the direct observation of cargo protein transport. We developed a new optically synchronized cargo release method called retention using the dark state of LOV2 (RudLOV). This innovative technique offers three exceptional control capabilities: spatial, temporal, and quantitative control of cargo release. RudLOV uses illumination to trigger transport and detect cargo. Consequently, the selection of an appropriate laser and filter set for controlling the illumination and/or detection is crucial. The protocol presented here provides step-by-step guidelines for obtaining high-resolution live imaging data using RudLOV, thereby enabling researchers to investigate intracellular cargo transport with unprecedented precision and control.
Generation of Insulin-Producing Alpha TC1-6 Cells Using EpiCRISPR System for Targeted DNA Methylation
Diabetes lacks concrete curative strategies due to diverse aetiologies and, therefore, represents the perfect candidate for cell replacement therapy, since it is caused by either an absolute (type 1 diabetes) or relative (type 2 diabetes) defect in the insulin-producing beta cells of the pancreas. Pancreatic alpha cells are a promising source for transdifferentiation into insulin-producing cells as they share a common developmental origin with beta cells and exhibit a certain degree of cellular plasticity. Furthermore, impairment of glucagon signaling in diabetes leads to a marked increase in alpha cell mass, raising the possibility that such alpha cell hyperplasia provides an increased supply of alpha cells for their transdifferentiation into new beta cells.In this protocol, we used the modular epigenetic CRISPR/dCas9 toolbox for targeted DNA methylation (EpiCRISPR) and silencing of the Arx gene (Aristaless Related Homeobox, Arx), which is essential for the maintenance of alpha cell identity. Methylation-based silencing of Arx initiates the reprogramming of pancreatic alpha cells into insulin-producing cells. As a key novelty, this protocol provides a direct route for epigenetically induced transdifferentiation of mouse pancreatic alpha TC1-6 cells into insulin-producing cells and thereby confirms a proof of concept of reversible cellular epigenetic reprogramming in vitro. In addition, this streamlined workflow addresses the inherent challenges of transfecting clustered alpha TC1-6 cells by optimizing their dissociation into single-cell suspensions, thereby improving uptake and reproducibility.In summary, this approach for cell transdifferentiation involves precise epigenetic editing of a lineage-specific marker gene, thereby enabling direct lineage conversion in a safe and versatile strategy to generate insulin-producing cells by epigenetic reprogramming. In contrast to approaches that rely on viral vectors or permanent genome editing, this method reduces the risk of off-target effects and immunogenic responses while ensuring reproducibility. The combination of efficiency and precision makes it a valuable tool to advance regenerative approaches for diabetes therapy and to explore the epigenetic regulation of cell identity.
Immunology
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.
Identification and Sorting of Adipose Inflammatory and Metabolically Activated Macrophages in Diet-Induced Obesity
Adipose tissue macrophages (ATMs) critically influence obesity-induced inflammation and metabolic dysfunction. Recent studies identified distinct ATM subsets characterized by markers such as CD11c, CD9, and Trem2, associated with pro-inflammatory and metabolically activated states. This protocol outlines a detailed, reproducible methodology for isolating, characterizing, and sorting these ATM subsets from murine epididymal white adipose tissue (eWAT) using multicolor flow cytometry. Key steps include stromal vascular fraction (SVF) isolation, immunophenotyping, sequential gating strategies, and fluorescence-activated cell sorting (FACS) for downstream gene expression analysis. The protocol was validated in diet-induced obese (DIO) mice treated with the IRE1 RNase inhibitor STF-083010, demonstrating its utility for studying ATMs in the context of obesity and metabolic disease.
Molecular Biology
Ribozyme-Mediated Knockdown of lncRNA Gene Expression in Drosophila
Long noncoding RNAs (lncRNAs) are increasingly understood to play important roles in cell biology, development, and disease, though the vast majority of annotated lncRNAs have yet to be functionally characterized. Disrupting lncRNAs is often challenging owing to their tolerance for mutations (e.g., single-nucleotide polymorphisms and short indels) along with the limitations of other genetic knockdown strategies such as RNA interference (RNAi). Here, we describe a protocol to achieve robust knockdown of lncRNAs in the fruit fly Drosophila using a self-cleaving ribozyme. The 111-bp ribozyme cassette, which consists of the N79 hammerhead ribozyme flanked by flexible linker sequences, is inserted into transcript regions of lncRNA genes using CRISPR/Cas9-mediated homology-directed repair (HDR). The fluorescent eye transformation marker is then removed using a piggyBac transposase, leaving no other modifications at the lncRNA locus save the ribozyme cassette insertion. When transcribed as part of the lncRNA, the ribozyme folds and catalyzes its own self-cleavage, resulting in two RNA cleavage fragments. The efficacy of lncRNA knockdown is then evaluated using reverse transcription quantitative PCR (RT-qPCR) and single-molecule RNA fluorescence in situ hybridization (smFISH). This approach has resulted in efficient knockdown of both nuclear and cytoplasmic lncRNAs in Drosophila, with knockdown of steady-state RNA levels in 3' cleavage fragments typically exceeding 90% and no evidence of off-target effects. The method can also be applied to protein-coding genes in order to knock down specific mRNA isoforms. Thus, self-cleaving ribozymes are a valuable addition to the genetic toolkit in Drosophila.
Efficient circRNA Detection Using the Processive Reverse Transcriptase uMRT
Circular RNAs (circRNAs) are covalently closed RNA molecules known for their increased stability compared to linear RNAs. Synthetic circRNAs are being developed as RNA therapeutics, while natural circRNAs are being investigated for their biological roles in eukaryotes and their potential as disease biomarkers. Consequently, the accurate detection and validation of circRNAs is crucial for advancements in both fundamental RNA research and biotechnological applications. Common methods for circRNA validation involve RT-PCR using divergent primers, followed by sequencing across the circRNA junction. However, most described methods are high-throughput approaches that require time-consuming RNA processing steps, and they are unable to detect highly structured circRNAs. Additionally, methods for low-throughput sequencing of small circRNAs (® (uMRT), a highly processive reverse transcriptase. Unlike other reverse transcriptases, uMRT can reverse-transcribe large, structured circRNAs of varying sizes, at ambient temperatures, enabling sequencing of the resulting concatemeric amplicons generated by RT-PCR and other methods. Using this method, we sequenced circRNAs containing highly structured internal ribosome entry sites commonly utilized in synthetic circRNAs, natural circRNAs containing repetitive elements, and small circRNAs, all without the need for cloning. With this new platform, we offer a protocol for the precise detection of nearly any circRNA species.
SunTag-Based Single-Molecule Translation Imaging in Caenorhabditis elegans
Translation is a key step in decoding the genetic information stored in DNA. Regulation of translation is an important step in gene expression control and is essential for healthy organismal development and behavior. Despite the importance of translation regulation, its impact and dynamics remain only partially understood. One reason is the lack of methods that enable the real-time visualization of translation in the context of multicellular organisms. To overcome this critical gap, microscopy-based methods that allow visualization of translation on single mRNAs in living cells and animals have been developed. A powerful approach is the SunTag system, which enables real-time imaging of nascent peptide synthesis with high spatial and temporal resolution. This protocol describes the implementation and use of the SunTag translation imaging system in the small round worm Caenorhabditis elegans. The protocol provides details on how to design, carry out, and interpret experiments to image translation dynamics of an mRNA of interest in a cell type of choice of living C. elegans. The ability to image translation live enables better understanding of translation and reveals the mechanisms underlying the dynamics of cell type–specific and subcellular localization of translation in development.
Plant Science
Effective Gene Silencing in Plants by Synthetic Trans-Acting siRNAs Derived From Minimal Precursors
Synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are 21-nucleotide small RNAs designed to induce highly specific and efficient gene silencing in plants. Traditional approaches rely on the transgenic expression of ~1 kb TAS precursors, which limits their use in non-model species, under strict GMO regulations, and in size-constrained expression or delivery systems. This protocol describes a rapid workflow for the design, assembly, and delivery of syn-tasiRNAs derived from much shorter precursors, referred to as minimal precursors. The pipeline includes in silico design of highly specific syn-tasiRNA sequences, cloning of minimal precursors into plant expression or potato virus X (PVX)-based viral vectors through Golden Gate or Gibson assembly, and delivery to plants through Agrobacterium-mediated expression or by spraying crude extracts containing recombinant PVX expressing the minimal precursors. These methodologies make syn-tasiRNA-based tools more accessible and broadly applicable for plant research and biotechnology across diverse species and experimental contexts.
Direct Plant Regeneration From Immature Male Inflorescence of Banana (Musa spp.)
Banana (Musa spp.) is a globally important horticultural crop that faces significant challenges from pests and diseases, which threaten yield and long-term sustainability. The efficient production of clean, disease-free planting material is essential for both commercial plantations and small-holder systems. This paper presents a rapid and reproducible protocol for direct plant regeneration from immature male inflorescences of banana. The method involves surface sterilization of immature male flowers, longitudinal dissection, and culture on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP), enabling direct shoot regeneration from floral meristems without an intermediate regenerable callus phase. This approach offers several advantages over traditional embryogenic cell suspension (ECS) methods, including simplified sterilization, high regeneration efficiency, and scalability. The protocol was successfully applied to multiple banana cultivars, including Cavendish (AAA) and Lady Finger (AAB), achieving 100% shoot regeneration efficiency with plantlet production within 6–8 months. This protocol provides a reliable and efficient alternative for rapid mass propagation of banana plants, supporting sustainable production and research applications.
Stem Cell
Rapid and Simplified Induction of Spinal Motor Neurons From Human Induced Pluripotent Stem Cells
Human induced pluripotent stem cell (hiPSC)-derived motor neurons (MNs) provide a critical source for the study of motor neuron diseases (MNDs), which has been hindered by the lack of appropriate disease models for many years. Although many spinal MN differentiation protocols have been established by mimicking in vivo neurogenesis using extrinsic signaling molecules, substantial variations in the duration and efficiency persist due to inconsistencies in concentrations, timing, and delivery methods of these molecules. Here, we present an efficient monolayer culture differentiation strategy that enables the generation of enriched CHAT+ spinal MNs (sMNs) in 18 days and functional sMNs exhibiting extensive network activities, as confirmed by multielectrode array (MEA), within 28 days. Therefore, this optimized MN differentiation protocol facilitates the production of mature sMNs for MND research, high-throughput drug screening, and potential cell replacement therapies.