Rab10 phosphorylation at Thr73 (pRab10) is a well-established readout of the kinase LRRK2, a protein whose mutations are associated with Parkinson’s disease. Here, we present a protocol for high-content quantification of endogenous pRab10-positive vesicles in primary astrocyte-enriched cultures using the Operetta CLS system. We describe steps for cell culture, immunofluorescence, image acquisition, and quantitative analysis using Harmony software. This protocol is applicable to other LRRK2-expressing cells and offers a scalable platform for quantitative studies of LRRK2 physiological and pathological activity.
Temporal regulation of cyclic adenosine monophosphate (cAMP) using photoactivated adenylyl cyclase (bPAC) is critical in research; however, commercial optogenetic systems are costly. Here, we present a protocol for optogenetic control of cAMP in human telencephalic organoids using an easy-to-build Arduino-driven light-emitting diode (LED) stimulation system. We describe steps for assembling the system, writing code for LED regulation, and preparing induced pluripotent stem (iPS) cells for optogenetic experiments. We then detail procedures for optogenetic activation of cAMP to control cell-fate decisions.For complete information on the generation and use of this protocol, please refer to Shimada et al.1
Persistent ductular reaction is a hallmark of chronic liver injury, characterized by the proliferation and pro-inflammatory activity of reactive biliary epithelial cells. Here, we present a protocol for modeling biliary cell activation in human precision-cut liver slices. We describe steps for collecting samples, generating, and culturing precision-cut liver slices, and activating biliary cells ex vivo while preserving native architecture and multicellular interactions. Biliary cell activation can be quantified by EdU incorporation, supporting mechanistic studies and translational drug-screening applications.For complete details on the use and execution of this protocol, please refer to Anfuso et al.1
Budding yeast is a model organism to study evolutionarily conserved biological processes, including plasma membrane (PM) repair. Here, we present a protocol to induce localized PM and cell wall damage during live-cell imaging. We describe steps for revival from frozen stock, liquid culture, inducing laser damage, and live-cell imaging. We then detail procedures for fluorescence-signal quantification and statistical analysis. This protocol enables the study of localized PM and cell wall repair mechanisms in budding yeast.
Presentation of taste solutions is required for investigating the detection and processing of gustatory information. These stimuli are also widely used to motivate animals in behavioral tasks. Here, we present a protocol for surgically implanting an intraoral cannula in mice, enabling direct and precise delivery of fluids for passive stimulation of oral sensory systems (e.g., gustation, somatosensation, and nociception) and associated limbic pathways. We then detail procedures for controlled solution delivery.For complete details on the use and execution of this protocol, please refer to Park and Lee.1
Tumors release soluble factors that reach tumor-draining lymph nodes (TDLNs), where they induce remodeling already at the pre-metastatic stage and alter the local proteomic landscape. Here, we describe a protocol for isolating interstitial fluid from primary tumors and TDLNs in murine breast cancer models to study extracellular protein composition while preserving tissue viability and integrity. We outline key steps for tissue preparation, fluid extraction, and downstream bioinformatic analysis to support the characterization of tumor-associated changes in TDLNs.For complete details on the use and execution of this protocol, please refer to Mattavelli et al.1
Rice grassy stunt virus (RGSV) causes a severe viral disease in rice and is transmitted by brown planthoppers (BPHs) (Nilaparvata lugens Stål). Here, we present a protocol for propagating RGSV and establishing controlled infection in rice plants. We describe steps for preparing rice seedlings, maintaining and propagating BPH colonies, generating viruliferous BPHs, and performing genotype-separated inoculation. This protocol provides a standardized framework for BPH-mediated RGSV inoculation and helps reduce vector-associated bias in infection assays across rice genotypes.For complete details on the use and execution of this protocol, please refer to Yang et al.1
Systematic elucidation of microtubule (MT) luminal proteins provides insights into MT biology and its molecular regulation. Here, we present a protocol for screening and validating these proteins using proximity labeling, mass spectrometry, fluorescence imaging and western blot (WB). We describe steps for constructing a biotin ligase knock-in cell line and performing BioID screening. We detail how to perform fluorescence imaging and WB to validate the MT luminal proteins. This protocol enables systematic identification to advance research on MT luminal proteins.For complete details on the use and execution of this protocol, please refer to Shao et al.1
Dried blood spots (DBS), most notably employed in newborn screening, present a valuable opportunity to analyze whole blood via a stable and simplified sample collection. DBS are generally used to evaluate one class of compounds at a time. Herein, we present a protocol to semi-quantitatively evaluate metabolites and lipids from the same DBS using a multi-omics approach. We describe steps for extracting both metabolites and lipids from the paper-based sample and analyzing them via liquid chromatography-tandem mass spectrometry.For complete details on the use and execution of this protocol, please refer to Li et al.1
Polar metabolites are challenging to analyze using common reverse-phase chromatography. However, these molecules are most essential for deciphering biological phenotypes. Here, we present a protocol for polar metabolite analysis based on anion-exchange chromatography coupled with high-resolution mass spectrometry (AEC-HRMS). We outline preparation techniques for common biological matrices, instrumental setup for untargeted metabolomics, including sample analysis and essential data treatment.
Polygenic prediction is often less accurate in admixed populations due to limited representation in training data. Here, we present a protocol for building a polygenic prediction model using admixed populations from the All of Us program. We describe steps for variant extraction, phasing, local ancestry inference, and construction of polygenic risk score. This protocol may also serve as a useful reference for researchers aiming to conduct large-scale analysis in the All of Us Research Workbench cost-effectively.For complete details on the use and execution of the protocol, please refer to Zhou et al.1
Here, we present a dynamic combinatorial library-versus-proteome activity-based protein profiling (DCL-ABPP) workflow for high-throughput covalent ligand discovery. We describe steps for integrating a dynamic combinatorial library with proteome-wide mass spectrometry to generate and screen hundreds of ligands in situ, eliminating the need for pre-synthesis. We detail procedures for two complementary modes: competitive screening, which identifies enzyme inhibitors (e.g., serine hydrolases) through family-wide probe competition; and direct screening, which maps covalent ligand binding sites on cysteines.For complete details on the use and execution of this protocol, please refer to Huang et al.1
Extrusion-based bioprinting is a promising approach for developing novel clinical therapies for heart failure patients. Here, we present a protocol for bioprinting personalized human-scale cardiac patches incorporating human cardiac spheroids in a 1% silk fibroin/4% alginate/8% gelatin hydrogel. We describe steps for extrusion 3D bioprinting using a temperature-controlled hydrogel bioink. We also detail adjusting print parameters for optimal results. Although prepared for a specific experimental setup, the techniques described can be applied to other temperature-controlled biomaterials or extrusion 3D bioprinters.For complete details on the use and execution of this protocol, please refer to Roche et al.,1,2 Sharma et al.,3 and Vettori et al.4
Oocyte degeneration is a critical and persistent limitation of intracytoplasmic sperm injection (ICSI). Here, we present assisted sperm fusion insemination (ASFI), a protocol for fertilizing human oocytes using zona pellucida-bound sperm (ZP-sperm) to minimize oocyte degeneration while preserving cellular integrity. We describe steps for co-culturing immature or degenerated oocytes with sperm, harvesting ZP-sperm, and facilitating their attachment to and fusion with oocytes. This protocol uses standard ICSI equipment and provides a reproducible approach for minimizing the mechanical stress during fertilization.For complete details on the use and execution of this protocol, please refer to Hatakeyama et al.1
Current T cell receptor (TCR) sequencing approaches have inherent technical limitations, and low-primer-bias techniques for quantitative genomic-level repertoire analysis remain limited. Here, we present high-throughput genome-wide translocation sequencing-based TCR sequencing (HTGTS-TCR-seq) as a genomic DNA-based protocol for low-bias analysis of TCR diversity. We describe steps for DNA fragmentation, LAM-PCR enrichment, streptavidin bead capture, adapter ligation, and PCR for library construction. This protocol enables quantitative profiling of TCR rearrangement products at the genomic DNA level.For complete details on the use and execution of this protocol, please refer to Luo et al.1
Here, we present an optimized methylation context-sensitive enzyme ddRAD (MCSeEd) library-preparation protocol for methylation analysis in CG, CHG, and CHH sequence contexts from plant samples that yield low-quality or partially degraded genomic DNA. We describe steps for quadruple restriction-enzyme digestion and adapter ligation, double size selection, freeze-and-squeeze gel purification, and bead-based cleanup. We then detail a two-step PCR enrichment procedure to generate Illumina-compatible epigenomic libraries.
Bone marrow plasma cells maintain durable antibody responses, but their tissue localization and morphology are difficult to quantify in situ. Here, we present a protocol for isotype-resolved 3D imaging and quantification of bone marrow plasma cells in murine femurs. We describe steps for fixation, optimal cutting temperature (OCT) coumpound embedding, longitudinal opening of mouse femurs, whole-mount immunostaining, optical clearing, and confocal 2D/3D imaging. We detail an Imaris-based workflow for reproducible single-cell segmentation, isotype-resolved counting, and the extraction of morphological features from defined regions of interest.
Here, we present a protocol for isolating extracellular vesicles (EVs) from the conditioned medium of human mesenchymal stromal cells (MSCs) and characterizing them using multiple modalities. We describe steps for enriching EVs by ultrafiltration and size-exclusion chromatography. We then detail procedures for imaging flow cytometry, transmission electron microscopy, nanoparticle tracking analysis, and the extraction of EV protein and RNA content. This workflow enables an integrated assessment of MSC-derived EVs to inform their use in diagnostic or therapeutic applications.
Tumor necrosis factor (TNF) is a key mediator of inflammation, acting as a proinflammatory cytokine with context-dependent dual roles. Here, we describe detailed assays to investigate key downstream signaling pathways of TNF involved in either cell death induction or NF-κB-mediated pro-survival signaling via the TNFR1 axis. We describe steps for target cell and reagent preparation, cell stimulation for cell death induction, and detection of activated intracellular signaling proteins by flow cytometry.For complete details on the use and execution of this protocol, please refer to Unmuth et al.1
DNA modifications are central to carcinogenesis, chemotherapy drug action, neurodegeneration, and aging. Here, we present a protocol for click-code-seq, a next-generation sequencing technique for genome-wide single-nucleotide-resolution mapping of major types of DNA modifications: oxidized guanines and abasic sites. We describe steps for genomic DNA extraction and library preparation, including enzymatic modification conversion and click-chemistry-based tagging. We then detail procedures for data analysis using the associated computational pipeline Click-pipe.For complete details on the use and execution of this protocol, please refer to Takhaveev et al.1
