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Current Issue in 2026
Volume: 16, Issue: 5
Biochemistry
Purification of the Active-State G Protein-Coupled Receptor ADGRL4 for Cryo-Electron Microscopy Using a Modular Tag System and a Tethered mini-Gq
ADGRL4 is an adhesion G protein-coupled receptor (aGPCR) implicated in tumour progression in multiple malignancies. We recently determined the first cryo-EM structure of active-state ADGRL4, revealing its weak coupling to the heterotrimeric G protein Gq and providing insights into its activation mechanism. Here, we describe a complete modular workflow for purifying active-state ADGRL4 over 2–3 days using a multifunctional tagging strategy incorporating multiple orthogonal detection, purification, and cleavage tags at the N-terminus as well as a tethered mini-Gq at the C-terminus. This configuration enhanced receptor cell-surface expression and stability and allowed different purification strategies to be tested during the development of the purification protocol. Although developed and optimised for ADGRL4, this approach is readily transferable to other weakly coupling aGPCRs or GPCRs where complex stability is a limiting factor for structural analysis.
Bioinformatics and Computational Biology
Machine Learning-Assisted Quantification of Organelle Abundance
Organelle abundance is a key microscopic readout of organelle formation and, in many cases, function. Quantification of organelle abundance using confocal microscopy requires estimating their area based on the fluorescence intensity of compartment-specific markers. This analysis usually depends on a user-defined intensity threshold to distinguish organelle regions from the surrounding cytoplasm, which introduces potential bias and variability. To address this issue, we present a machine learning–assisted algorithm that allows for the quantification of organelle density using the open-source Fiji platform and WEKA segmentation. Our method enables the automated quantification of organelle number, area, and density by learning from training data. This standardizes threshold selection and minimizes user intervention. We demonstrate the utility of this approach for both membrane and non-membrane organelles, such as peroxisomes, lipid droplets, and stress granules, in human cells and whole fish samples.
High-Resolution Quantification of Two-Way Nanobody Synergy Using Automated Liquid Handling and Computational Modeling
Evaluating single-domain antibody cooperativity is essential for developing potent, escape-resistant antiviral biologics. Here, we present a protocol that reproducibly quantifies functional synergy between neutralizing nanobody pairs in standardized viral infectivity assays. Controlled automated liquid handling prepares two-dimensional concentration matrices, minimizing pipetting variance and systematic error. Neutralization data are fitted using quantitative models that independently estimate potency, cooperativity, and efficacy to distinguish additive, synergistic, and antagonistic effects between nanobody pairs. Replicated measurements enable statistically interpretable parameter estimates, supporting robust evaluation of combinatorial nanobody therapeutics with commonly available equipment and open-source analysis tools. This framework is broadly applicable to assessing cooperative effects among other classes of binding or inhibitory molecules, facilitating systematic discovery of synergistic combinations.
Spatial Proteomics Using S4P
Spatial proteomics enables the mapping of protein distribution within tissues, which is crucial for understanding cellular functions in their native context. While spatial transcriptomics has seen rapid advancement, spatial proteomics faces challenges due to protein non-amplifiability and mass spectrometry sensitivity limitations. This protocol describes a sparse sampling strategy for spatial proteomics (S4P) that combines multi-angle tissue strip microdissection with deep learning–based image reconstruction. The method achieves whole-tissue slice coverage with significantly reduced sampling requirements, enabling mapping of over 9,000 proteins in mouse brain tissue at 525 μm resolution within 200 h of mass spectrometry time. Key advantages include reduced sample processing time, deep proteome coverage, and applicability to centimeter-sized tissue samples.
Cancer Biology
Non-Enzymatic Isolation of Cancer-Associated Fibroblasts From Human Prostate Tumor Explants
Prostate carcinoma (PCa) progression is strongly influenced by the surrounding tumor microenvironment, where cancer-associated fibroblasts (CAFs) represent the most abundant and functionally relevant stromal population. Despite their importance, the lack of stable cell lines representing CAF phenotypes limits the study of stromal–tumor interactions. To address this limitation, we provide an optimized protocol for isolating CAFs from fresh human PCa biopsies based on a mechanical procedure exploiting the specific CAF ability to migrate out from the tumor explants. This approach preserves tissue architecture and maintains CAF viability and phenotype. The resulting ex vivo CAF cultures provide a suitable model to investigate CAF biology within the tumor microenvironment.
Cell Biology
Quantifying Lysosomal Degradation of Extracellular Proteins With a Fluorescent Protein-Based Internalization Assay
Endocytosis is an essential membrane transport mechanism that is indispensable for the maintenance of life. It is responsible for the selective internalization and subsequent degradation or recycling of specific extracellular proteins and nutrients, thereby facilitating cellular nutrient supply, modulation of receptor signaling, and clearance of foreign substances. However, methods for the quantitative analysis of lysosomal degradation of extracellular proteins via endocytosis remain limited. This protocol describes a method for purifying the protein-of-interest (POI)–red fluorescent protein (RFP)–green fluorescent protein (GFP) fusion protein, which is modified with specific mammalian cell glycans or other modifications, from the conditioned medium of mammalian cell cultures. Subsequently, the protocol details a quantitative approach for evaluating its internalization and lysosomal degradation within cells using the RFP–GFP tandem fluorescent reporter. Following the addition of POI-RFP-GFP to the medium, cells can be subjected to cell biological assays, such as flow cytometry, as well as biochemical analyses, such as immunoblotting. This protocol is broadly applicable to studies of the internalization of extracellular proteins.
Obtaining Chondroprogenitors (Articular Cartilage-Derived Cells) via Explant Methodology
Obtaining articular cartilage-derived cells (chondroprogenitors) by explant methodology is a reliable approach for isolating migratory progenitor cells that retain strong chondrogenic potential. This method allows cells to emerge naturally from small cartilage fragments without enzymatic digestion. The procedure consists of plating cartilage explants on a plastic surface with culture medium, from which cells subsequently migrate and adhere to the substrate. Compared with enzymatic isolation, the explant approach minimizes cellular stress and better reproduces the physiological microenvironment of cartilage tissue. This protocol can be applied to both osteoarthritic and non-osteoarthritic samples, enabling comparative studies on disease-related phenotypic differences. Overall, this technique offers a reproducible, straightforward, and minimally invasive strategy for obtaining functional chondroprogenitor cells suitable for cartilage regeneration research.
A Rapid and High-Recovery Extracellular Vesicle (EVs) Isolation Technique from Blood Samples
Extracellular vesicles (EVs) circulating in blood serve as non-invasive “liquid biopsies,” carrying molecular cargo that reflects the physiological and pathological state of distant cells. Their analysis is crucial for understanding disease mechanisms and discovering novel biomarkers. Clinically, blood EVs hold significant promise for early disease diagnosis, prognostic assessment, and monitoring treatment response in diverse areas such as organ transplantation, cancer, and neurological disorders. Current EV isolation techniques, beyond ultracentrifugation, include size exclusion chromatography (separation by size for high purity) and immunoaffinity capture (using antibodies for high specificity). Here, we present a simplified, rapid, and reproducible method for isolating EVs from small-volume blood samples. This protocol consistently yields a concentrated EV pellet covering 50–300 nm EVs, amenable to direct downstream analysis. Developed and validated in our laboratory using human, porcine, and murine blood samples, this method has proven instrumental in identifying EV-based biomarkers for predicting outcomes related to organ transplantation. The protocol’s adaptability and reliance on readily prepared, cost-effective reagents further enhance its utility. This scalable approach can be further integrated with subsequent purification or enrichment steps to optimize sample preparation for protein and nucleic acid assays.
Microbiology
Dynamic Mapping of RNA-Binding Proteins During Bacillus subtilis Sporulation Using Orthogonal Organic Phase Separation
RNA-binding proteins (RBPs) have pleiotropic roles in modulating the physiology of both eukaryotic and prokaryotic cells, enabling them to adapt to environmental variations. The importance of RBPs has led to the development of a variety of methods aiming to identify them. However, most of these approaches have primarily been implemented and optimized in eukaryotic systems. To both uncover novel RBPs involved in Bacillus subtilis sporulation and capture their RNA-binding ability dynamically, we adapted the orthogonal organic phase separation technique (OOPS), which had previously been used in Escherichia coli to reveal its RNA-binding proteome (RBPome). We optimized the UV cross-linking process used to stabilize RNA–protein interactions in vivo and the bacterial lysis process to overcome the robust cell wall of Gram-positive sporulating cells. RNA–protein complexes are then recovered after phase separation steps using guanidinium thiocyanate–phenol–chloroform, and RNA-associated proteins are identified and label-free-quantified by liquid chromatography–mass spectrometry. Collecting samples at various time points during sporulation further enables tracking the dynamics of the RBPome. In addition to being applicable to bacteria and requiring minimal starting material, this method has provided a comprehensive map of the RBPome during sporulation, refining the roles of known factors and revealing new players.
A Standardized Culture Medium for Comparative Drug Efficacy Evaluation Across Plasmodium and Babesia Species
The discovery of broad-spectrum antiparasitic agents relies on the ability to evaluate drug efficacy under harmonized in vitro conditions across related species. However, current drug screening pipelines for intraerythrocytic parasites are constrained by the use of species-specific media with distinct nutrient compositions and serum sources, which hinder direct comparison of compound potency. To address this gap, we describe a unified erythrocytic culture system based on DMEM/F12 supplemented with 20% fetal bovine serum (DFS20), which supports robust asexual growth of multiple Plasmodium falciparum strains (3D7, Dd2, HB3, V1/S), Babesia duncani, Babesia divergens (Rouen 87), and Babesia MO1. Parasite proliferation and morphology in DFS20 are comparable to those observed in established species-specific media such as RPMI-1640 for P. falciparum and B. divergens and HL-1/Claycomb/DMEM/F12/SFM for B. duncani, while eliminating reliance on undefined or discontinued proprietary components. Importantly, this standardized medium enables cross-species growth inhibition assays for direct comparison of drug efficacy under identical conditions. Using this platform, we recently screened dihydrotriazines and biguanides targeting the conserved DHFR-TS enzymes and identified potent antifolate candidates with broad-spectrum activity against Babesia and Plasmodium species. For B. duncani, which is uniquely supported by both a continuous in vitro human erythrocyte culture system and a lethal in vivo mouse infection model, integration with the in-culture and in-mouse (ICIM) pipeline enables systematic validation of pharmacodynamics, pharmacokinetics, resistance, and toxicity. This unified DFS20-based system establishes a scalable and reproducible protocol for harmonized drug efficacy evaluation across intraerythrocytic parasites and provides a foundation for the development and prioritization of pan-antiparasitic therapies.
Framework for Analyzing the Anti-biofilm and Anti-virulence Activities of Fatty Acids from Hermetia illucens Larvae Targeting Multidrug-Resistant Klebsiella pneumoniae
The emergence of antimicrobial resistance and the persistence of Klebsiella pneumoniae biofilms represent significant challenges to public health. Hermetia illucens (HI) larvae are considered a sustainable reservoir of novel bioactive compounds. This protocol details a method for extracting fatty acids from HI larvae fat (AWME3 fraction) and studying their effects on multidrug-resistant and hypervirulent Klebsiella pneumoniae strains. Effects are evaluated by crystal violet and ethidium bromide uptake assays, motility assays (swimming, swarming, and twitching), minimal biofilm inhibitory and eradication concentration tests (MBIC/MBEC) for single, mixed, and mature biofilms, light, fluorescence, and scanning electron microscopy imaging, and microbial adhesion to solvents (MATS). This protocol offers a reliable methodology for evaluating the anti-biofilm and anti-virulence properties of natural compounds.
Molecular Biology
aGPCR-HEK: A Stable High-Expression Inducible Mammalian Cell Expression System for Adhesion GPCR Structural Biology Applications
ADGRL4 is an adhesion G protein–coupled receptor (aGPCR) implicated in multiple tumours. In our experience, conventional insect cell-based baculovirus expression systems have not yielded sufficient correctly folded ADGRL4 protein for purification and cryo-electron microscopy (cryo-EM) analysis. Here, we describe aGPCR-HEK, a six-week protocol that establishes stable tetracycline-inducible mammalian HEK293S GnTI- TetR cell lines expressing N-terminally HA- and GFP-tagged aGPCRs. The method comprises lentiviral production in Lenti-X 293T cells, transduction of target adherent HEK293S GnTI- TetR cells, flow cytometry enrichment of uninduced GFP-positive cells displaying leaky expression, adaptation to suspension culture, and large-scale tetracycline induction and harvesting of cells for downstream purification and cryo-EM. The system yields reproducible, milligram-scale quantities of folded aGPCR suitable for structural and biochemical studies.
Employing Tribe to Study RNA Interactions of Ataxin-2 in Drosophila S2 Cells
RNA-binding protein (RBP)–RNA interactions are fundamental for gene regulation and cellular homeostasis. Ataxin-2 is an RBP that has been shown to play an instrumental role in pathophysiological processes by binding to mRNA. Methods such as RNA immunoprecipitation (RIP), cross-linking immunoprecipitation (CLIP), and their variants can be used to study the interactions between Ataxin-2 and its targets, although their high sample requirements and labor-intensive workflows can limit their widespread use. RNA editing-based approaches, such as targets of RBPs identified by editing (TRIBE), provide effective alternatives. TRIBE enables transcriptome-wide identification of RBP targets by inducing site-specific adenosine-to-inosine (A-to-I) editing, which is subsequently detected through high-throughput RNA sequencing in both in vivo and in vitro systems. Compared to in vivo models, cell lines offer a rapid and flexible experimental design. Drosophila S2 cells are a commonly used insect cell line to investigate RNA–protein dynamics and serve as a versatile platform for studying RBP function. Here, we describe a protocol used for identifying RNA targets of Ataxin-2, a versatile RBP involved in post-transcriptional and translational regulation, in S2 cells using TRIBE. This method allows rapid, efficient, and reliable identification of Ataxin-2-associated RNA targets and can be readily applied to other RBPs.
Selective Isolation of TOP3B•mRNA Covalent Intermediates Using Denaturing Oligo-dT Pulldown
The deletion and mutation of Topoisomerase 3β (TOP3B) is linked to multiple neurological disorders and is the only known topoisomerase that is also catalytically active on RNA in vitro and in cells. Uniquely, TOP3B is primarily localized to the cytoplasm, binds to open reading frames of mRNA, and regulates mRNA stability and translation in a transcript-specific manner. A common approach for studying TOP3B activity in cells is immunodetection of TOP3B•RNA covalent intermediates after bulk RNA isolation. However, in this approach, the RNA species is unknown and is not selective for the major TOP3B substrate, mRNA. In this protocol, we describe a recently developed and optimized protocol for capturing TOP3B•mRNA covalent intermediates using oligo-dT isolation of mRNA under protein-denaturing conditions. Covalent intermediates are then detected by a dual membrane slot blotting strategy with nitrocellulose and positively charged nylon membranes. Nitrocellulose membrane-bound TOP3B•mRNA covalent intermediates are analyzed by immunodetection, and nylon membrane-bound free mRNA is stained with methylene blue. The protocol detailed below has been validated with wildtype and mutant 3xFLAG-tagged TOP3B expressed in Neuro2A cells, with additional optimization for slot blotting using recombinant EGFP.
Neuroscience
Reconstruction of Axonal Projections of Single Neurons Using PointTree
The morphology of single-neuron axonal projections is critical for deciphering neural circuitry and information flow in the brain. Yet, manually reconstructing these complex, long-range projections from high-throughput whole-brain imaging data remains an exceptionally labor-intensive and time-consuming task. Here, we developed a points assignment-based method for axonal reconstruction, named PointTree. PointTree enables the precise identification of the individual axons from densely packed axonal population using a minimal information flow tree model to suppress the snowball effect of reconstruction errors. In this protocol, we have elaborated on how to configure the required environment for PointTree software, prepare suitable data for it, and run the software. This protocol can assist neuroscience researchers in more easily and rapidly obtaining the reconstruction results of neuronal axons.
Combining Suction-Pipette Spectral Identification With Single-Cell RT-PCR to Make Differential Analyses of Amphibian Red and Green Rods
Amphibian retinas contain “green” rods, which are rod-shaped photoreceptors with a cone-type visual pigment. These rods are considered a potentially transitional photoreceptor type, but their phototransduction cascade’s molecular composition has remained uncertain. Here, we present a streamlined electrophysiology-molecular workflow that enables the rapid spectral identification, physical capture, and targeted single-cell reverse transcription-polymerase chain reaction (RT-PCR) of individual amphibian photoreceptors. After suction-pipette spectral screening under alternating red and green illumination, electrophysiologically identified cells are isolated and processed directly for reverse transcription and PCR. Coupling real-time functional phenotyping with sensitive molecular profiling provides a practical tool for resolving photoreceptor molecular heterogeneity and investigating evolutionary transitions between rod and cone phenotypes.
Plant Science
Simple Induction and Detection of Anthocyanins in Arabidopsis thaliana: A Tool for Mutant Screening and Metabolic Analysis
Anthocyanins are specialized flavonoid pigments that play critical roles in plant coloration, photoprotection, and responses to environmental stress. Arabidopsis thaliana serves as a valuable genetic model for dissecting anthocyanin biosynthesis and regulatory networks. Conventional methods for anthocyanin quantification, such as crude spectrophotometric assays, often compromise pigment integrity, yield inconsistent results, and provide limited information on compound composition. Here, we describe a simple, reproducible, and high-fidelity protocol for the induction, extraction, quantification, and chromatographic profiling of anthocyanins in Arabidopsis thaliana seedlings. The workflow employs well-defined anthocyanin-inductive conditions (AIC), methanol/formic acid extraction, lyophilization for dry-weight normalization, and dual quantification via spectrophotometry and High-performance liquid chromatography with diode-array detection (HPLC-DAD) analysis. This protocol enables accurate comparison between wild-type and mutant genotypes, facilitating both mutant screening and metabolic pathway analysis. The approach minimizes pigment degradation, enhances reproducibility across replicates, and offers a robust tool for research in plant metabolism, stress physiology, and flavonoid biochemistry.
Stem Cell
Development, Expansion, and Histological Characterization of Patient-Derived Liver Organoids for Drug Screening and Disease Modeling
Organoids are self-organizing 3D tissues representing an innovative technology with interesting implications and potential for the study of tumor biology. They can be developed from fine-needle biopsies or resection material from healthy or tumor tissues. Patient-derived organoids are able to retain most of the histological characteristics, the expression profile, and the genomic landscape of the corresponding primary tissues, making them suitable for translational studies and for the identification of molecular alterations in the field of personalized medicine. Here, we describe a detailed protocol for the preparation and in vitro expansion of tumor and non-tumor organoids from surgical resections or needle biopsies of patients with hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), enabling subsequent testing of small-molecule VDAC1 antagonists at different doses. In parallel, we developed a hepatic steatosis model by treating healthy liver organoids with oleic acid, recapitulating key features of lipid accumulation and metabolic dysfunction in vitro. This protocol enables the generation of patient-derived liver organoids that preserve the histological and molecular characteristics of their original tissue, providing a robust and versatile platform for translational studies, personalized drug testing, and the exploration of novel therapeutic strategies targeting tumor metabolism.