Analysis Basic Protocol Research Articles

RCPA: An Open-Source R Package for Data Processing, Differential Analysis, Consensus Pathway Analysis, and Visualization.

Identifying impacted pathways is important because it provides insights into the biology underlying conditions beyond the detection of differentially expressed genes. Because of the importance of such analysis, more than 100 pathway analysis methods have been developed thus far. Despite the availability of many methods, it is challenging for biomedical researchers to learn and properly perform pathway analysis. First, the sheer number of methods makes it challenging to learn and choose the correct method for a given experiment. Second, computational methods require users to be savvy with coding syntax, and comfortable with command-line environments, areas that are unfamiliar to most life scientists. Third, as learning tools and computational methods are typically implemented only for a few species (i.e., human and some model organisms), it is difficult to perform pathway analysis on other species that are not included in many of the current pathway analysis tools. Finally, existing pathway tools do not allow researchers to combine, compare, and contrast the results of different methods and experiments for both hypothesis testing and analysis purposes. To address these challenges, we developed an open-source R package for Consensus Pathway Analysis (RCPA) that allows researchers to conveniently: (1) download and process data from NCBI GEO; (2) perform differential analysis using established techniques developed for both microarray and sequencing data; (3) perform both gene set enrichment, as well as topology-based pathway analysis using different methods that seek to answer different research hypotheses; (4) combine methods and datasets to find consensus results; and (5) visualize analysis results and explore significantly impacted pathways across multiple analyses. This protocol provides many example code snippets with detailed explanations and supports the analysis of more than 1000 species, two pathway databases, three differential analysis techniques, eight pathway analysis tools, six meta-analysis methods, and two consensus analysis techniques. The package is freely available on the CRAN repository. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Processing Affymetrix microarrays Basic Protocol 2: Processing Agilent microarrays Support Protocol: Processing RNA sequencing (RNA-Seq) data Basic Protocol 3: Differential analysis of microarray data (Affymetrix and Agilent) Basic Protocol 4: Differential analysis of RNA-Seq data Basic Protocol 5: Gene set enrichment analysis Basic Protocol 6: Topology-based (TB) pathway analysis Basic Protocol 7: Data integration and visualization.

RNA-Seq Data Analysis: A Practical Guide for Model and Non-Model Organisms.

RNA sequencing (RNA-seq) has emerged as a powerful tool for assessing genome-wide gene expression, revolutionizing various fields of biology. However, analyzing large RNA-seq datasets can be challenging, especially for students or researchers lacking bioinformatics experience. To address these challenges, we present a comprehensive guide to provide step-by-step workflows for analyzing RNA-seq data, from raw reads to functional enrichment analysis, starting with considerations for experimental design. This is designed to aid students and researchers working with any organism, irrespective of whether an assembled genome is available. Within this guide, we employ various recognized bioinformatics tools to navigate the landscape of RNA-seq analysis and discuss the advantages and disadvantages of different tools for the same task. Our protocol focuses on clarity, reproducibility, and practicality to enable users to navigate the complexities of RNA-seq data analysis easily and gain valuable biological insights from the datasets. Additionally, all scripts and a sample dataset are available in a GitHub repository to facilitate the implementation of the analysis pipeline. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Analysis of data from a model plant with an available reference genome Basic Protocol 2: Gene ontology enrichment analysis Basic Protocol 3: De novo assembly of data from non-model plants.

Turning Trash Into Treasure: A Simple Protocol for Human Mesenchymal Stromal Cell Isolation From Used Bone Marrow Collection Kits.

The therapeutic potential of mesenchymal stromal cells (MSCs) has been extensively investigated in both preclinical and clinical settings. Recent years have witnessed the emergence of numerous isolation protocols and culture techniques, ranging from the selection of subpopulations to preserve stemness to preconditioning strategies aimed at enhancing therapeutic efficacy, tailored to the specific tissue source. In this protocol, we present a straightforward and cost-effective method for isolating human MSCs (hMSCs) from discarded bone marrow collection kits (comprising bag and filter systems) originally intended for removing impurities and unwanted cellular debris from the collected bone marrow aspirate, ensuring the purity of the stem cell population during stem cell transplantation. Utilizing basic laboratory equipment, we demonstrate the isolation of hMSCs, highlighting the expression of specific surface antigens, and multilineage differentiation into adipogenic, osteogenic, and chondrogenic lineages in vitro. This sustainable and resource-efficient approach not only contributes to reducing medical waste but also holds promise for advancing regenerative medicine applications. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Isolation of human mesenchymal stromal cells from bone marrow collection kits Basic Protocol 2: Culture of human mesenchymal stromal cells Basic Protocol 3: Characterization of human mesenchymal stromal cells with flow cytometry analysis Basic Protocol 4: Characterization of human mesenchymal stromal cells with multilineage differentiation under in vitro conditions.

Data-Independent Acquisition and Label-Free Quantification for Quantitative Proteomics Analysis of Human Cerebrospinal Fluid.

This article presents a practical guide to mass spectrometry-based data-independent acquisition and label-free quantification for proteomics analysis applied to cerebrospinal fluid, offering a robust and scalable approach to probing the proteomic composition of the central nervous system. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Cerebrospinal fluid sample collection and preparation for mass spectrometry analysis Basic Protocol 2: Mass spectrometry sample analysis with data-independent acquisition Support Protocol: Data-dependent mass spectrometry and spectral library construction Basic Protocol 3: Analysis of mass spectrometry data.

High-Dimensional Methods of Single-Cell Microglial Profiling to Enhance Understanding of Neuropathological Disease.

Microglia are the innate myeloid cells of the central nervous system (CNS) parenchyma, functionally implicated in almost every defined neuroinflammatory and neurodegenerative disorder. Current understanding of disease pathogenesis for many neuropathologies is limited and/or lacks reliable diagnostic markers, vaccines, and treatments. With the increasing aging of society and rise in neurogenerative diseases, improving our understanding of their pathogenesis is essential. Analysis of microglia from murine disease models provides an investigative tool to unravel disease processes. In many neuropathologies, bone-marrow-derived monocytes are recruited to the CNS, adopting a phenotype similar to that of microglia. This significantly confounds the accurate identification of cell-type-specific functions and downstream therapeutic targeting. The increased capacity to analyze more phenotypic markers using spectral-cytometry-based technologies allows improved separation of microglia from monocyte-derived cells. Full-spectrum profiling enables enhanced marker resolution, time-efficient analysis of >40 fluorescence parameters, and extraction of cellular autofluorescence parameters. Coupling this system with additional cytometric technologies, including cell sorting and high-parameter imaging, can improve the understanding of microglial phenotypes in disease. To this end, we provide detailed, step-by-step protocols for the analysis of murine brain tissue by high-parameter ex vivo cytometric analysis using the Aurora spectral cytometer (Cytek), including best practices for unmixing and autofluorescence extraction, cell sorting for single-cell RNA analysis, and imaging mass cytometry. Together, this provides a toolkit for researchers to comprehensively investigate microglial disease processes at protein, RNA, and spatial levels for the identification of therapeutic targets in neuropathology. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Processing the mouse brain into a single-cell suspension for microglia isolation Basic Protocol 2: Staining single-cell mouse brain suspensions for microglial phenotyping by spectral cytometry Basic Protocol 3: Flow cytometric sorting of mouse microglia for ex vivo analysis Basic Protocol 4: Processing the mouse brain for imaging mass cytometry for spatial microglia analysis.

Analytical Workflows for Single-Cell Multiomic Data Using the BD Rhapsody Platform.

The conversion of raw sequencing reads to biologically relevant data in high-throughput single-cell RNA sequencing experiments is a complex and involved process. Drawing meaning from thousands of individual cells to provide biological insight requires ensuring not only that the data are of the highest quality but also that the signal can be separated from noise. In this article, we describe a detailed analytical workflow, including six pipelines, that allows high-quality data analysis in single-cell multiomics. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Image analysis Basic Protocol 2: Sequencing quality control and generation of a gene expression matrix Basic Protocol 3: Gene expression matrix data pre-processing and analysis Basic Protocol 4: Advanced analysis Basic Protocol 5: Conversion to flow cytometry standard (FCS) format Basic Protocol 6: Visualization using graphical interfaces.

MSeqDR Quick-Mitome (QM): Combining Phenotype-Guided Variant Interpretation and Machine Learning Classifiers to Aid Primary Mitochondrial Disease Genetic Diagnosis.

The international Mitochondrial Disease Sequence Data Resource Consortium (MSeqDR) Quick-Mitome (QM) is a web-based platform enabling automated variant interpretation of whole-exome sequencing (WES) datasets for the genetic diagnosis of primary mitochondrial diseases (PMD). Designed specifically to address the unique dual genome nature of PMD etiologies, QM includes features for both nuclear and mitochondrial DNA (mtDNA) genome analysis. QM requires VCF variant lists, HPO ID clinical phenotypes, and pedigree files for multiple-sample VCF inputs. QM maps phenotypes to HPO terms before analysis. QM analysis requires 2 to 20 min for 100,000 variants on an 8-vCPU AWS server using Exomiser's "PASS_ONLY" mode for nuclear variants. QM ranks variants based on allele frequency, phenotype-gene association, functional impact, and inheritance mode. Variants are further annotated with multiple data sources such as OMIM, ClinVar, dbNSFP, gnoMAD, MITOMAP, and MSeqDR. In addition to standard Exomiser results, QM generates an Analysis Report and QM Integrated Report with add-on mtDNA-specific analyses, including haplogroup prediction with Phy-Mer, heteroplasmy calculation, and mvTool annotations. We developed the Mitochondrial Disease Variant (MDV) classifier using XGBoost to predict variant pathogenicity for PMD. The MDV classifier was trained on >120 features and performance benchmarking showed that it correctly classified >98% of nuclear gene variants as being pathogenic or benign, and predicted PMD-causing variants with 94% precision. The MSeqDR QM server is an open-access resource for phenotype-driven dual-genome analyses for PMD diagnosis by the global mitochondrial disease community. It is publicly available for non-commercial, non-clinical research use at https://mseqdr.org/quickmitome.php. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Standardizing clinical phenotypes into human phenotype ontology (HPO) terms as the phenotype input for Quick-Mitome (QM) Basic Protocol 2: Prepare the pedigree input for multiple-sample VCF Basic Protocol 3: Quick-Mitome (QM) analysis Basic Protocol 4: Reviewing and understanding the QM Integrated Report and Analysis Report.

High-Content Screening of Synaptic Density Modulators in Primary Neuronal Cultures.

The synapse, which represents the structural and functional basis of neuronal communication, is one of the first elements affected in several neurodegenerative diseases. To better understand the potential role of gene expression in synapse loss, we developed an original high-content screening (HCS) model capable of quantitatively assessing the impact of gene silencing on synaptic density. Our approach is based on a model of primary neuronal cultures (PNCs) from the neonatal rat hippocampus, whose mature synapses are visualized by the relative localization of the presynaptic protein Synaptophysin with the postsynaptic protein Homer1. The heterogeneity of PNCs and the small sizes of the synaptic structures pose technical challenges associated with the level of automation necessary for HCS studies. We overcame these technical challenges, automated the processes of image analysis and data analysis, and carried out tests under real-world conditions to demonstrate the robustness of the model developed. In this article, we describe the screening of a custom library of 198 shRNAs in PNCs in the 384-well plate format. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culture of primary hippocampal rat neurons in 384-well plates Basic Protocol 2: Lentiviral shRNA transduction of primary neuronal culture in 384-well plates Basic Protocol 3: Immunostaining of the neuronal network and synaptic markers in 384-well plates Basic Protocol 4: Image acquisition using a high-throughput reader Basic Protocol 5: Image segmentation and analysis Basic Protocol 6: Synaptic density analysis.

A Protocol for Culture and Characterization of Human Induced Pluripotent Stem Cells After Induction.

Human induced pluripotent stem cells (hiPSCs) are characterized by unlimited self-renewal and the capability to differentiate into all three germ layers, with the potential to further differentiate into all types of cells and tissues. Human iPSCs retain all genetic information from their original donors and can be developed into disease models to study disease pathophysiology, identify disease phenotypes and biomarkers, and evaluate therapeutic efficacy and toxicity for drug development. Human iPSCs can also be used to develop cell therapies and regenerative medicine. In the last decade, the technologies for hiPSC generation and differentiation have advanced rapidly. Human iPSC culture and propagation are tedious and require careful handling. High-quality hiPSCs are necessary for downstream applications. The methods, techniques, and skills for hiPSC maintenance and characterization are very different from those for immortalized cell lines. It can be a challenge for new laboratory staff, and sometimes even for experienced staff, to properly culture and maintain the high quality of these cells. Here, we describe a comprehensive set of protocols for hiPSC propagation under chemically defined and feeder-free culture conditions. These step-by-step protocols describe in detail all the reagents and experimental procedures needed to culture hiPSCs. The protocols also describe experimental methods for hiPSC characterization, including immunofluorescence staining and flow cytometric analysis with a panel of pluripotency markers, a teratoma formation assay for validation of in vivo pluripotency, and detection of Sendai virus to ensure elimination of the viral vectors. These protocols have been successfully used in our laboratory for hiPSC expansion and propagation, and this article provide a useful reference guide for laboratory staff to work on hiPSC culture. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Propagation and cryopreservation of hiPSC cultures Basic Protocol 2: Recovery of cryopreserved hiPSCs Basic Protocol 3: Validation of pluripotency markers via immunocytochemical analysis Alternate Protocol: Determination of the expression of pluripotency markers via flow cytometry analysis Basic Protocol 4: Assessment of pluripotency via in vivo teratoma formation assay Basic Protocol 5: Confirmation of Sendai viral vector clearance via RT-PCR.

Mouse Models for Atopic Dermatitis.

Atopic dermatitis (AD) is a multifactorial disease with underlying barrier disruption and altered microbial flora, resulting in dry skin and eczematous inflammation with persistent pruritis. Mouse models have been heavily used to investigate AD pathophysiology. Among various AD mouse models, AD-like inflammation induced by topical calcipotriol, a vitamin D3 analog referred to as MC903 in experimental settings, is a versatile model that can be applied to any strain of mice, which can be used for immunologic and morphologic studies. Herein, we provide basic protocols for the topical application of MC903 and approaches to assess phenotypes. After inducing AD-like inflammation, the skin is harvested for flow cytometry analysis, as well as for histologic and immunofluorescence microscopy analyses. The combination of these approaches enables accurate characterization of the degree of inflammation, type of inflammatory infiltrate, and localization of immune infiltrates. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Application of MC903 and gross phenotype assessment Basic Protocol 2: Processing skin for flow cytometry analysis Support Protocol: Skin immune cell surface staining and flow cytometry analysis Basic Protocol 3: Harvesting skin for histologic analysis Basic Protocol 4: Immunofluorescence staining to identify immune cell infiltrates.

Glia Cell Morphology Analysis Using the Fiji GliaMorph Toolkit.

Glial cells are the support cells of the nervous system. Glial cells typically have elaborate morphologies that facilitate close contacts with neighboring neurons, synapses, and the vasculature. In the retina, Müller glia (MG) are the principal glial cell type that supports neuronal function by providing a myriad of supportive functions via intricate cell morphologies and precise contacts. Thus, complex glial morphology is critical for glial function, but remains challenging to resolve at a sub-cellular level or reproducibly quantify in complex tissues. To address this issue, we developed GliaMorph as a Fiji-based macro toolkit that allows 3D glial cell morphology analysis in the developing and mature retina. As GliaMorph is implemented in a modular fashion, here we present guides to (a) setup of GliaMorph, (b) data understanding in 3D, including z-axis intensity decay and signal-to-noise ratio, (c) pre-processing data to enhance image quality, (d) performing and examining image segmentation, and (e) 3D quantification of MG features, including apicobasal texture analysis. To allow easier application, GliaMorph tools are supported with graphical user interfaces where appropriate, and example data are publicly available to facilitate adoption. Further, GliaMorph can be modified to meet users' morphological analysis needs for other glial or neuronal shapes. Finally, this article provides users with an in-depth understanding of data requirements and the workflow of GliaMorph. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Download and installation of GliaMorph components including example data Basic Protocol 2: Understanding data properties and quality 3D-essential for subsequent analysis and capturing data property issues early Basic Protocol 3: Pre-processing AiryScan microscopy data for analysis Alternate Protocol: Pre-processing confocal microscopy data for analysis Basic Protocol 4: Segmentation of glial cells Basic Protocol 5: 3D quantification of glial cell morphology.

SP3-FAIMS-Enabled High-Throughput Quantitative Profiling of the Cysteinome.

Cysteine-directed chemoproteomic profiling methods yield high-throughput inventories of redox-sensitive and ligandable cysteine residues and therefore are enabling techniques for functional biology and drug discovery. However, the cumbersome nature of many sample preparation workflows, the requirements for large amounts of input material, and the modest yields of labeled peptides are limitations that hinder most chemoproteomics studies. Here, we report an optimized chemoproteomic sample-preparation workflow that combines enhanced peptide labeling with single-pot, solid-phase-enhanced sample preparation (SP3) to improve the recovery of biotinylated peptides, even from small samples. We further tailor our SP3 method to specifically probe the redox proteome, which showcases the utility of the SP3 platform in multistep sample-preparation workflows. By implementing a customized workflow in the FragPipe computational pipeline, we achieve accurate MS1-based quantification, including for peptides containing multiple cysteine residues. Collectively these innovations enable enhanced high-throughput quantitative analysis of the cysteinome. This article includes detailed protocols for cysteine labeling with isotopically labeled iodoacetamide alkyne probes, biotinylation with CuAAC, sample cleanup with SP3, enrichment of cysteines with NeutrAvidin agarose beads, LC-FAIMS-MS/MS analysis, and FragPipe-IonQuant analysis. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Labeling of cysteines in human proteome and SP3-based sample cleanup Alternate Protocol 1: Labeling of cysteines in human proteome, SP3-based sample cleanup, and enrichment of cysteines for isoTOP-ABPP analysis Alternate Protocol 2: Labeling of cysteines in human proteome and SP3-based sample cleanup for redox proteome analysis Basic Protocol 2: Peptide-level cysteine enrichment Basic Protocol 3: LC-FAIMS-MS/MS analysis Basic Protocol 4: FragPipe data analysis.

Triplet-Primed PCR Assays for Accurate Screening of FMR1 CGG Repeat Expansion and Genotype Verification.

Fragile X syndrome and other fragile X-associated disorders are caused by the full-mutation (>200 copies) and premutation (55 to 200 copies) expansion, respectively, of the CGG short tandem repeat in the fragile X messenger ribonucleoprotein 1 (FMR1) gene. Clinical diagnostic laboratories use Southern blot analysis and polymerase chain reaction (PCR)-based tests to detect and/or size the FMR1 CGG repeats. The development of sensitive and high-throughput triplet-primed PCR (TP-PCR) assays has diminished the need to subject all samples to Southern blot analysis, which is both labor- and time-intensive. In this article, we describe two direct TP-PCR (dTP-PCR) assays for the detection of FMR1 CGG repeat expansions. We outline a protocol that is based on melting curve analysis of dTP-PCR amplicons for a rapid and cost-effective first-tier screening and identification of individuals with premutation and full-mutation expansions. We also describe a protocol that employs capillary electrophoresis to resolve the dTP-PCR amplicon fragments and to estimate the repeat sizes of normal (5 to 44 copies), intermediate (45 to 54 copies), and premutation alleles, as well as to detect full mutations and determine the structure of the FMR1 alleles. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Direct triplet-primed PCR master mix preparation and amplification of the FMR1 CGG repeat locus for melting curve analysis Basic Protocol 2: Melting curve analysis of direct triplet-primed PCR amplicons on the Rotor-Gene Q MD × 5plex high-resolution melt platform Alternate Protocol: Melting curve analysis of direct triplet-primed PCR amplicons on the LightCycler 480 system Basic Protocol 3: Generation of direct triplet-primed PCR melting curve analysis profiles Basic Protocol 4: Direct triplet-primed PCR master mix preparation and amplification of the FMR1 CGG repeat locus for capillary electrophoresis Basic Protocol 5: Generation of control FMR1 plasmids for direct triplet-primed PCR melting curve analysis Basic Protocol 6: Sanger sequencing assay to verify FMR1 CGG repeat size and structure of plasmid DNA controls.

Gel-Based and Gel-Free Phosphoproteomics to Measure and Characterize Mitochondrial Phosphoproteins.

The mitochondrion is a key intracellular organelle regulating metabolic processes, oxidative stress, energy production, calcium homeostasis, and cell survival. Protein phosphorylation plays an important role in regulating mitochondrial functions and cellular signaling pathways. Dysregulation of protein phosphorylation status can cause protein malfunction and abnormal signal transduction, leading to organ dysfunction and disease. Investigating the mitochondrial phosphoproteins is therefore crucial to better understand the molecular and pathogenic mechanisms of many metabolic disorders. Conventional analyses of phosphoproteins, for instance, via western blotting, can be done only for proteins for which specific antibodies to their phosphorylated forms are available. Moreover, such an approach is not suitable for large-scale study of phosphoproteins. Currently, proteomics represents an important tool for large-scale analysis of proteins and their post-translational modifications, including phosphorylation. Here, we provide step-by-step protocols for the proteomics analysis of mitochondrial phosphoproteins (the phosphoproteome), using renal tubular cells as an example. These protocols include methods to effectively isolate mitochondria and to validate the efficacy of mitochondrial enrichment as well as its purity. We also provide detailed protocols for performing both gel-based and gel-free phosphoproteome analyses. The gel-based analysis involves two-dimensional gel electrophoresis and phosphoprotein-specific staining, followed by protein identification via mass spectrometry, whereas the gel-free approach is based on in-solution mass spectrometric identification of specific phosphorylation sites and residues. In all, these approaches allow large-scale analyses of mitochondrial phosphoproteins that can be applied to other cells and tissues of interest. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Mitochondrial isolation/purification from renal tubular cells Support Protocol: Validation of enrichment efficacy and purity of mitochondrial isolation Basic Protocol 2: Gel-based phosphoproteome analysis Basic Protocol 3: Gel-free phosphoproteome analysis.

Flow Virometry for Characterizing the Size, Concentration, and Surface Antigens of Viruses.

Application of flow cytometry principles for the analysis of viruses has been referred to as flow virometry (FVM). FVM is a multiparametric, high-throughput, and sensitive technique that allows viral particles to be detected, quantified, and characterized based on the biophysical properties of the virus and the expression of proteins on their surface. More specifically, by calibrating the flow cytometer with reference materials, it is possible to measure the concentration of intact viral particles in a sample, the abundance of a target antigen on the surface of the virus, and the relative diameter of the virus. Here, we describe a comprehensive overview of procedures used to stain, detect, and quantify viral and host-derived proteins located on the surface of retroviruses. These outlined techniques can be applied for the rapid phenotypic characterization of retroviruses, other enveloped viruses, and generally most viruses at the single-particle level through the direct staining of viruses collected from the supernatant of infected cells, without the need for enrichment or purification. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Virus production Basic Protocol 2: Instrument setup, standardization, and quality control for fluorescence quantification Basic Protocol 3: Flow virometry analysis Basic Protocol 4: Viral surface antigen staining and fluorescence quantification Support Protocol: Determination of the optimal antibody concentration for virus staining Basic Protocol 5: Gain configuration optimization.

Establishing an In Vitro 3D Spheroid Model to Study Medulloblastoma Drug Response and Tumor Dissemination

Medulloblastoma is the most common malignant pediatric brain tumor. Current treatment involves surgery, chemotherapy, and craniospinal radiotherapy, and these are associated with a significant reduction in quality of life. Metastatic dissemination at diagnosis is found in up to 30% of medulloblastoma cases and, alongside therapy resistance, is a significant feature in determining poor outcome. Development of new therapeutic approaches requires models where drug resistance and migration can be readily quantified and that are representative of patient disease. 3D medulloblastoma (3D-MB) spheroids are a simple yet effective means of bridging the gap between 2D culture and in vivo methods, providing users with highly reproducible in vitro models that more accurately recapitulate tumor morphology, drug response, and migration from a tumor mass. Unlike other cancer types, medulloblastoma spheroids fail to grow in their different standard cell culture media; instead, each cell line requires the same stem cell-enriching conditions. This requirement, however, has the advantage that it allows direct comparison of growth and response between cell lines in the absence of any potential media bias. In addition, spheroids can be used to model the initial stages of metastatic dissemination, something that cannot be achieved in 2D culture, providing insight into key changes occurring in migratory cells. Here, we provide protocols that detail the initial generation and maintenance of 3D-MB spheroids from sonic-hedgehog, Group 3, and Group 4 medulloblastoma subgroups, as well as describing functional assays to study drug response and cell migration across hyaluronan matrices, which represent the extracellular matrix backbone of the brain parenchyma. Through application of these simple yet highly representative models, it will be possible to test novel therapeutics targeting metastasis and drug resistance, as well as to develop insights into the mechanistic processes driving relapse in this malignant pediatric brain tumor. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation and maintenance of 3D medulloblastoma (3D-MB) spheroids Support Protocol 1: Measuring spheroid size for coefficient-of-variation analysis Basic Protocol 2: Assessing drug response in 3D-MB spheroids Support Protocol 2: 384-well 3D-MB spheroid generation Basic Protocol 3: Immunohistochemical staining of 3D-MB spheroids Basic Protocol 4: Modeling metastatic dissemination using 3D-MB migration models Support Protocol 3: RNA extraction from 3D-MB spheroids.

Decellularized Extracellular Matrix for Cell Biology.

The extracellular matrix (ECM) is an architecture that supports the cells in our bodies and regulates various cell functions. The ECM is composed of many proteins and carbohydrates, and these molecules activate various intracellular signaling pathways orchestrated to decide cell fates. Therefore, it is not enough to study the role of single ECM molecules to understand the roles of the ECM in the regulation of cell functions; it is necessary to understand how the ECM, as an assembly of various molecules, regulates cell functions as a whole. For this purpose, in vitro ECM models mimicking native ECM are required. Here, a decellularization technique is presented to reconstitute native ECM in vitro. In this article, methods for preparing decellularized ECM (dECM) are described for use in tumor and stem cell biology. Additionally, a method for confirmation of decellularization and a dECM modification method are described. These dECM types will be useful for comprehensive studies of ECM roles. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of in vitro extracellular matrix (ECM) models mimicking native ECM in different malignant tumor tissues Basic Protocol 2: Preparation of in vitro ECM models mimicking native ECM surrounding myoblasts differentiating into myotubes at each myogenic stage Support Protocol 1: Confirmation of myogenic stages by myogenic stages by myogenic gene expression analysis Basic Protocol 3: Confirmation of cell removal Basic Protocol 4: Reduction of chondroitin sulfate chains in cultured cell-derived decellularized ECM Support Protocol 2: Quantification of chondroitin sulfate chain amounts in the decellularized ECM.

Guide to the Larval Zebrafish-Aspergillus Infection Model.

The larval zebrafish is an increasingly popular host model for the study of Aspergillosis. The visual accessibility, genetic resources, small size, and ease of handling make zebrafish larvae compatible with higher-throughput investigation of fungal virulence and host resistance mechanisms. This article provides the protocols needed to prepare Aspergillus fumigatus spore inocula and use microinjection to infect the hindbrain ventricle of zebrafish larvae. Furthermore, we include protocols for analyzing host survival, immobilizing larvae for live imaging, and suggestions for image analysis. © 2021 Wiley Periodicals LLC. Support Protocol 1: Preparing Aspergillus spores Support Protocol 2: Dechorionating zebrafish embryos Support Protocol 3: Generating transparent larvae with 1-phenyl 2-thiourea (PTU) Basic Protocol 1: Hindbrain microinjection of zebrafish larvae with Aspergillus spores Basic Protocol 2: Survival analysis Basic Protocol 3: Multi-day imaging of infected larvae Alternate Protocol: Embedding larvae in low-melting-point agarose.

Drug-releasing Microspheres for Stem Cell Differentiation.

The ability of stem cells to differentiate into specialized cells make them a valuable tool for therapeutic applications. 3D bioprinting, a subset of additive manufacturing, uses bioinks composed of cells and biomaterials to create living tissues. The use of bioactive factors like small molecules and proteins can promote stem cell differentiation into the desired cell phenotypes for tissue regeneration. Small molecules can accelerate the process of regeneration in tissue engineering, maintain bioactivity in a biological environment, and minimize the costs associated with this process. Additionally, they can be encapsulated in specialized drug-delivery devices called microspheres for controlled release. Microspheres are small (1-1000 μm) spherical particles usually made from biodegradable and biocompatible polymers that can be loaded with drugs and other bioactive components. They can then be integrated into stem-cell-laden bioinks used to form bioprinted tissues, where they will release the encapsulated drug and promote differentiation of stem cells into the desired mature cell type. Microspheres can be widely used to encapsulate a broad range of therapeutic agents, including hydrophilic and hydrophobic small molecule drugs, DNA, and proteins. The release of encapsulated molecules occurs through degradation and erosion of the polymer matrix. This article provides detailed protocols for fabricating and sterilizing drug-releasing microspheres made from poly-ε-caprolactone, a promising biodegradable polymer often used for controlled drug delivery due to its biocompatibility and biodegradation kinetics. Additional protocols describe characterization of the loading and size of microspheres as well as incorporation of microspheres into a fibrin-based bioink for 3D bioprinting. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Fabrication of drug-releasing PCL microspheres Support Protocol 1: Preparation of microspheres for determination of encapsulation efficiency by HPLC Support Protocol 2: Preparation of microspheres for SEM analysis Basic Protocol 2: Incorporation of microspheres into fibrin-based bioink.

High-Resolution Multi-Isotope Imaging Mass Spectrometry (MIMS) Imaging Applications in Stem Cell Biology.

Multi-isotope imaging mass spectrometry (MIMS) allows the measurement of turnover of molecules within intracellular compartments with a spatial resolution down to 30 nm. We use molecules enriched in stable isotopes administered to animals by diet or injection, or to cells through the culture medium. The stable isotopes used are, in general, 15 N, 13 C, 18 O, and 2 H. For stem cell studies, we essentially use 15 N-thymidine, 13 C-thymidine, and 81 Br from BrdU. This protocol describes the practical use of MIMS with specific reference to applications in stem cell research. This includes choice and administration of stable isotope label(s), sample preparation, best practice for high-resolution imaging, secondary ion mass spectrometry using the Cameca NanoSIMS 50L, and methods for robust statistical analysis of label incorporation in regions of interest (ROI). © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Stable isotope labeling of DNA in cultured cells Basic Protocol 2: Stable isotope labeling of DNA in animals Basic Protocol 3: Preparation of Si chips, the general sample support for NanoSIMS analysis Basic Protocol 4: Stable isotope analysis of DNA replication in single nuclei in a population of cells with NanoSIMS Basic Protocol 5: Data reduction and processing.