STAR Protocols

Protocol for the quantitative characterization of cell aggregates using an MRI setup maintaining optimal cultivation conditions The use of destructive biochemical assays and the preparation of histologic samples are routinely employed to monitor development and viability of 3D cell aggregates. Magnetic resonance imaging (MRI) offers a non-destructive, high-resolution alternative to histological analysis, enabling longitudinal assessment of cellular dynamics while preserving sample integrity. Here, we present a protocol for non-invasive MR imaging of cell spheroid clusters by creating an adequate imaging environment. We d...

Protocol for the quantitative characterization of cell aggregates using an MRI setup maintaining optimal cultivation conditions #protocol #starprotocols #cellpress

Protocol for detecting oral squamous cell carcinoma in histopathology images using the momentum contrast framework The detection of oral squamous cell carcinoma (OSCC) in histopathology images is crucial for improving diagnostic accuracy and patient outcomes. Here, we present a protocol for detecting OSCC in histopathology images using transfer learning. We describe steps for installing software and prerequisites, preparing datasets, and pretraining a model on images from various tissue types using the momentum contrast (MoCo) framework. We then detail procedures for evaluating the fine-tuned HistoMOCO model...

Protocol for detecting oral squamous cell carcinoma in histopathology images using the momentum contrast framework #protocol #starprotocols #cellpress

Protocol for decellularizing and delipidizing human pancreatic tissue into native hydrogel We present a protocol for transforming human pancreatic tissue into a decellularized and delipidized extracellular matrix (ECM) for hydrogel/scaffold applications. We describe steps for decellularization and delipidization using sodium deoxycholate and sequential ethanol, acetone, and hexane washes and removal of nucleic acids with benzonase. We then detail procedures for lyophilizing the ECM for long-term preservation and digesting the pancreatic ECM in pepsin-HCL to form a hydrogel. For comple...

Protocol for decellularizing and delipidizing human pancreatic tissue into native hydrogel #protocol #starprotocols #cellpress

Protocol to study adenosine release in the adult mouse hippocampus using a genetically encoded sensor, 2-photon live imaging, and fiber photometry Genetically encoded fluorescent sensors are powerful tools for tracking real-time neuromodulator dynamics. Here, we present a protocol to measure adenosine release using the G protein-coupled receptor activation-based adenosine (GRABAdo1.0m) sensor in the mouse hippocampus. We describe steps for stereotaxic surgery, including virus injection and optic fiber implantation, ex vivo two-photon live imaging, and in vivo fiber photometry. We then detail histological procedures for experimental validat...

Protocol to study adenosine release in the adult mouse hippocampus using a genetically encoded sensor, 2-photon live imaging, and fiber photometry #protocol #starprotocols #cellpress

Protocol to study internalization and localization dynamics of exogenously added proteins in detergent-permeabilized human cells Cell-permeabilization and resealing assays are essential tools for studying the localization and functional roles of exogenous factors in detergent-permeabilized cells. We describe a protocol for studying the internalization and localization of exogenous proteins in detergent-permeabilized human cells. It includes detailed steps for culturing, permeabilizing, and preparing ghost cells, followed by lysate incubation and fixation. Subsequent immunofluorescence staining and imaging allow visualizat...

Protocol to study internalization and localization dynamics of exogenously added proteins in detergent-permeabilized human cells #protocol #starprotocols #cellpress

Protocol for the recombinant expression and purification of the LSAM domain of human legumain in E. coli Expressing disulfide-rich proteins in E. coli is challenging due to incorrect bond formation. Here, we present a protocol for expressing the PC1pro-LSAM fusion protein in E. coli using the PC1 prodomain as a fusion tag and the legumain stabilization and activity modulation (LSAM) domain as a proof-of-concept target that was purified. We describe steps for characterizing the protein’s structural integrity through multiple biochemical and biophysical parameters like molecular weight, melting tempe...

Protocol for the recombinant expression and purification of the LSAM domain of human legumain in E. coli #protocol #starprotocols #cellpress

Protocol for deriving distance restraints from AlphaFold for use in solution NMR structure determination Artificial intelligence (AI) has revolutionized structural biology but must be applied reliably. Here, we present an approach for derivation of distance restraints from AlphaFold structure predictions to aid in automated nuclear Overhauser effect (NOE) assignment during solution NMR structure determination. We describe steps for selecting reliable AlphaFold structure predictions, determination of atom distances, and generation of high-confidence distance restraints. This protocol can expedite so...

Protocol for deriving distance restraints from AlphaFold for use in solution NMR structure determination #protocol #starprotocols #cellpress

Protocol to isolate stress granules in HeLa cells using fluorescence-activated non-membrane condensate isolation Various stress stimuli induce cytosolic stress granules (SGs) in mammalian cells, which are composed of RNA and RNA-binding proteins and have important physiological functions. Here, we present a protocol for isolating SGs using fluorescence-activated non-membrane condensate isolation (FANCI). We describe steps for seeding and stressing G3BP1-mCherry HeLa cells. We then detail procedures for purifying SGs by FANCI with flow cytometry. This protocol has potential application in studying SGs from ...

Protocol to isolate stress granules in HeLa cells using fluorescence-activated non-membrane condensate isolation #protocol #starprotocols #cellpress

Protocol for analyzing T cell and macrophage motility in a mouse pancreatic cancer spheroid model using light-sheet microscopy The interaction of macrophages and T cells is pivotal for shaping the immune response to cancer. Here, we present a protocol for investigating the co-culture of macrophages and T cells in a pancreatic cancer spheroid model. We describe steps for analyzing migration patterns by light-sheet microscopy, generating murine bone marrow-derived macrophages (BMDMs) and cytotoxic T lymphocytes (CTLs), and staining CTLs for image analysis. We then detail procedures for generating spheroids using a mouse p...

Protocol for analyzing T cell and macrophage motility in a mouse pancreatic cancer spheroid model using light-sheet microscopy #protocol #starprotocols #cellpress

Protocol for click labeling of HIV-1 envelope on amber-free virions prepared using genetic code expansion Site-specific fluorescent labeling of HIV-1 proteins enables direct visualization of their dynamics and trafficking. Here, we present a protocol to label HIV-1 envelope (Env) glycoproteins on virions using genetic code expansion and click chemistry. We describe steps for using HEK293T cells to produce amber-free HIV-1 carrying a noncanonical amino acid (ncAA) and for fluorophore conjugation to the ncAA. This protocol offers a practical way to apply amber suppression and click chemistry to track ...

Protocol for click labeling of HIV-1 envelope on amber-free virions prepared using genetic code expansion #protocol #starprotocols #cellpress

E3RC: A step-by-step computational protocol for exploring enhancer RNA expression and regulation using conventional RNA-seq data Here, we present a computational framework, E3RC, for exploring enhancer RNA (eRNA) expression and regulation using conventional RNA sequencing (RNA-seq) data. Using a public dataset as an example, we describe step-by-step guidelines for identifying eRNAs and characterizing their expressional changes and transcriptional regulation across multiple developmental stages of mouse preimplantation embryos. For complete details on the use and execution of this protocol, please refer to Yu et al.1

E3RC: A step-by-step computational protocol for exploring enhancer RNA expression and regulation using conventional RNA-seq data #protocol #starprotocols #cellpress

Protocol for developing a mouse model of post-primary pulmonary tuberculosis after hematogenous spread in native lungs and lung implants Here, we present a protocol for a mouse model for studying mechanisms of post-primary pulmonary tuberculosis (PTB) caused by virulent Mycobacterium tuberculosis (Mtb) using subcutaneous hock infection and lung tissue implantation. We describe steps for collagen instillation of lungs, lung and spleen implantation, preparation of Mtb for infection, and hock infection of mice. We then detail procedures for the perfusion of the lung and collection of organs, tissue processing, and histopathologic in...

Protocol for developing a mouse model of post-primary pulmonary tuberculosis after hematogenous spread in native lungs and lung implants #protocol #starprotocols #cellpress

Protocol for differentiating human pluripotent stem cells into midbrain organoids for targeted microinjection of viruses Midbrain organoids serve as a valuable model for studying brain development and disease. Here, we present a viral infection protocol for midbrain organoid models. We describe steps for human pluripotent stem cell (hPSC) differentiation into midbrain organoids; equipment preparation; and targeted virus microinjection into organoids to reduce viral load, minimize cytotoxicity, and preserve structural integrity. We then detail procedures for visualizing infected cells. By combining the virus with s...

Protocol for differentiating human pluripotent stem cells into midbrain organoids for targeted microinjection of viruses #protocol #starprotocols #cellpress

Protocol for processing and analyzing multiplexed images improves lymphatic cell identification and spatial architecture in human tissue Multiplexed images of human lymphatic tissue are extensively preprocessed before cell phenotyping and spatial analysis. Here, we present KINTSUGI (knowledge integration with new technologies: simplified user-guided image processing), a protocol designed to interactively engage the user in each processing step to ensure quality control. We describe steps for parameter tuning and batch processing of raw image data including illumination correction, stitching, deconvolution, 3D-2D conversion, regis...

Protocol for processing and analyzing multiplexed images improves lymphatic cell identification and spatial architecture in human tissue #protocol #starprotocols #cellpress

Protocol for assessing mitochondrial cholesterol transport and protein molten globule state in steroidogenic and nonsteroidogenic systems Steroid hormones are essential for the survival of all mammals for carbohydrate metabolism, stress management, and sexual reproduction. Here, we present a protocol for assessing mitochondrial cholesterol transport and protein molten globule state via measurement of pregnenolone or progesterone synthesis in steroidogenic and nonsteroidogenic cellular systems. We describe steps for cell culture, transfection, and measurement of steroidogenic activity from nonsteroidogenic cells. We then detail pro...

Protocol for assessing mitochondrial cholesterol transport and protein molten globule state in steroidogenic and nonsteroidogenic systems #protocol #starprotocols #cellpress

Protocol update to: High-throughput scNMT protocol for multiomics profiling of single cells from mouse brain and pancreatic organoids Single-cell nucleosome, methylome, and transcriptome (scNMT) sequencing is a recently developed method that allows multiomics profiling of single cells. In this scNMT protocol, we describe profiling of cells from mouse brain and pancreatic organoids, using liquid handling platforms to increase throughput from 96-well to 384-well plate format. Our approach miniaturizes reaction volumes and incorporates the latest Smart-seq3 protocol to obtain higher numbers of detected genes and genomic DNA (gDNA...

Protocol update to: High-throughput scNMT protocol for multiomics profiling of single cells from mouse brain and pancreatic organoids #protocol #starprotocols #cellpress

Protocol for visualization of ultrafine nuclear structures in cultured cells using protein retention expansion microscopy In the nucleus, multivalent interactions between DNA, RNA, and proteins form functional networks that drive nuclear metabolism, such as transcription and RNA processing. Here, we present a protocol to visualize the subnuclear distribution of nuclear proteins in cultured cells using protein retention expansion microscopy (proExM). We describe steps for immunostaining, sample expansion, sample bonding on glass coverslips, and imaging with confocal microscopy. This protocol is a reproducible and co...

Protocol for visualization of ultrafine nuclear structures in cultured cells using protein retention expansion microscopy #protocol #starprotocols #cellpress

Protocol for minicircle production for gene therapy without subsequent cleanup steps Current techniques for producing minicircles have low yields because of the cleanup steps required. Here, we present a protocol that can increase minicircle yield up to 10-fold by eliminating post-induction cleanup steps. We describe steps for bacterial culture, same-day arabinose induction, and DNA harvesting. This protocol is relevant for all minicircles produced from parental plasmids containing attB, attP, and 32 copies of Isce-I endonuclease sites in the minicircle-producing bacteria, ZYCY1...

Protocol for minicircle production for gene therapy without subsequent cleanup steps #protocol #starprotocols #cellpress

Protocol for directed differentiation of human induced pluripotent stem cells into thyroid follicular epithelial cells We provide a directed differentiation protocol deriving thyroid follicular cells (TFCs) from human induced pluripotent stem cells (iPSCs) or embryonic stem cells without exogenous transcription factors, recapitulating the sequence of developmental milestones of thyroid development. We describe a protocol for seeding iPSCs, inducing definitive and anterior foregut endoderm, followed by thyroid lineage specification via Fibroblast Growth Factor/Bone Morphogenetic Protein (FGF/BMP) signaling and su...

Protocol for directed differentiation of human induced pluripotent stem cells into thyroid follicular epithelial cells #protocol #starprotocols #cellpress

Protocol for direct reprogramming of canine fibroblasts to induced motor neurons Direct reprogramming of canine fibroblasts to induced motor neurons in vitro opens the door to molecular characterization, functional analysis, and therapeutic screening of canine age-related neurodegenerative diseases. Here, we present a protocol for generating canine induced motor neurons directly from primary dermal fibroblasts. We provide a step-by-step guide for isolating and maintaining primary dermal fibroblasts from dogs, obtaining a stock of reprogrammable cells, and performing the dire...

Protocol for direct reprogramming of canine fibroblasts to induced motor neurons #protocol #starprotocols #cellpress

Protocol for generation, quantification, and phenotyping of brain metastases in preclinical mouse models The field of brain metastasis is rapidly expanding, yet no consensus exists on the most reliable quantification approach. We present a protocol for assessing metastatic burden in mice following intracarotid injection of tumor cells. We describe steps for surgical procedures, brain processing, cryo-sectioning, and slide preparation, followed by phenotypic characterization. We detail procedures for quantifying brain metastatic area using automated microscopy and semi-supervised image analysis. For...

Protocol for generation, quantification, and phenotyping of brain metastases in preclinical mouse models #protocol #starprotocols #cellpress

Protocol for detecting LDLR on the cell surface of primary mouse hepatocytes using cell-surface biotinylation Here, we present a protocol for selective biotinylation, solubilization, and enrichment of plasma membrane proteins from primary mouse hepatocytes. We describe steps for injecting C57/BL6 mice via the tail vein with adeno-associated virus subtype 8 (AAV8) to specifically knock down the target gene in hepatocytes. We then detail procedures for labeling surface proteins with EZ-LINK Sulfo-NHS-LC-Biotin and enriching low-density lipoprotein receptor (LDLR) with immunoprecipitation. Finally, we outl...

Protocol for detecting LDLR on the cell surface of primary mouse hepatocytes using cell-surface biotinylation #protocol #starprotocols #cellpress

Protocol to quantify compound retention in Drosophila melanogaster for the pharmacokinetic study of orally administered compounds Drosophila melanogaster is a valuable tool for intestinal function research. Here, we present a compound retention assay (CORE) to quantify the orally administered compounds that were absorbed, metabolized, or accumulated in the tissues (for example, fat) of D. melanogaster. We describe steps for preparing flies and media, compound administration, and sample collection. We then detail procedures for determining concentrations and calculating CORE values. This protocol circumvents the limitations...

Protocol to quantify compound retention in Drosophila melanogaster for the pharmacokinetic study of orally administered compounds #protocol #starprotocols #cellpress

Protocol to generate species-specific astrocyte-conditioned medium for human organoid neuron maturation Brain organoids have emerged as promising models for neuroscience research; however, their utility is often limited by inadequate maturation. Here, we present a protocol for enhancing the maturation of human 2D and 3D neural cultures using astrocyte-conditioned medium (ACM). We outline the steps for isolating primary mouse astrocytes, culturing both mouse and human astrocytes, and preparing ACM. Additionally, we provide detailed procedures for quality control and downstream applications of ACM d...

Protocol to generate species-specific astrocyte-conditioned medium for human organoid neuron maturation #protocol #starprotocols #cellpress

Protocol to distinguish pre-mRNA from mRNA in RNA-protein interaction studies Transcriptome-wide studies on interactions between RNA-binding proteins (RBPs) and protein-coding RNAs in general preclude interpretations regarding RBP preference for binding to the more abundant mRNA over the less abundant pre-mRNA. Here, we present a protocol to determine the binding preference of the RBP tristetraprolin (TTP, Zfp36) for pre-mRNA versus mRNA. We describe steps for the identification and quantitation of intronic and exonic fragments in RNA bound to TTP. This protocol can poten...

Protocol to distinguish pre-mRNA from mRNA in RNA-protein interaction studies #protocol #starprotocols #cellpress

Protocol for the establishment and characterization of South African patient-derived intestinal organoids Patient-derived organoids are valuable for modeling disease pathogenesis, therapeutic screening, and advancing personalized medicine. Here, we present a protocol for generating and characterizing intestinal organoids from South African patients, using both healthy and cancerous tissues. We describe steps for generating organoid cultures, quantitative reverse-transcription polymerase chain reaction (RT-qPCR), immunofluorescence microscopy, and histological staining. Notably, we provide a detailed...

Protocol for the establishment and characterization of South African patient-derived intestinal organoids #protocol #starprotocols #cellpress

Protocol for detecting interactions between intrinsically disordered proteins and long DNA substrates by electrophoretic mobility shift assay Intrinsically disordered regions (IDRs) of proteins leverage their structural flexibility to play important roles in numerous cellular processes including molecular recognition. Many IDRs interact with DNA, and characterizing these interactions is crucial for understanding their biological impact. Here, we present a protocol for the in vitro detection of IDR-DNA interactions using an electrophoretic mobility shift assay. We describe a radioactive-free procedure using long DNA substrates and defi...

Protocol for detecting interactions between intrinsically disordered proteins and long DNA substrates by electrophoretic mobility shift assay #protocol #starprotocols #cellpress

Protocol for tissue expansion microscopy for ultrastructure expansion of Xenopus embryos Tissue expansion microscopy (TissUExM) allows super-resolution imaging by physically expanding biological samples. Here, we present a protocol for ultrastructure expansion microscopy of Xenopus embryos using TissUExM. We describe steps for tissue fixation, embedding, fluorescence labeling, and expansion. We demonstrate that our protocol provides enhanced resolution for studying subcellular structures, such as centrioles and cilia. This protocol has the potential to offer a broad range of applica...

Protocol for tissue expansion microscopy for ultrastructure expansion of Xenopus embryos #protocol #starprotocols #cellpress

Protocol for GCaMP expression and in vivo calcium imaging of medial prefrontal cortex neurons in freely behaving HIV-1 Tat transgenic mice Advancements in imaging techniques have enhanced our ability to study brain structure and function. Genetically encoded calcium indicators enable high-resolution visualization of neuronal activity at the cellular level. Here, we present a protocol for imaging medial prefrontal cortex (mPFC) neurons in a freely behaving neuroHIV mouse model. We describe steps for survival surgery preparation, craniotomy, durotomy, and adeno-associated virus (AAV) injection. We then detail procedures for implantin...

Protocol for GCaMP expression and in vivo calcium imaging of medial prefrontal cortex neurons in freely behaving HIV-1 Tat transgenic mice #protocol #starprotocols #cellpress

Protocol for differential analysis of pseudouridine modifications using nanopore DRS and unmodified transcriptome control In this protocol, we detect pseudouridine using nanopore direct RNA sequencing (DRS 002/004) and analyze dynamic changes to mRNA modifications in response to perturbation. We cover RNA extraction, poly(A) selection, DRS, unmodified transcriptome (in vitro transcribed [IVT]) library preparation, and knockdowns of writer proteins as critical controls. We detail preprocessing, psi-site identification, and differential analysis, quantifying the direction and magnitude of changes across cellular type...

Protocol for differential analysis of pseudouridine modifications using nanopore DRS and unmodified transcriptome control #protocol #starprotocols #cellpress