Cancers of the Nervous System

This video demonstrates the establishment of luciferase-tagged glioma stem cells via lentivirus-mediated gene delivery. Luciferase acts as a reporter gene and helps facilitate rapid drug screening experiments.

In this video, we describe a method highlighting the utility of bioluminescence assay for sensitive and rapid screening of drugs. These drugs can be tested alone or in combination to assess the synergism in their mode of action.

In this video, we perform the microglial phagocytosis assay for neuroblastoma using induced pluripotent stem cells. The assay helps assess the impairment of microglial function in neurodegenerative diseases.

This video describes the protocol to isolate single-nuclei from fresh frozen glioma tissue. The isolated single-nuclei can be used as specimens for sequencing techniques such as single-nuclei RNA (snRNA) sequencing and single-nuclei Assay for Transposase-Accessible Chromatin (snATAC) sequencing to obtain deeper insights into inter- and intra-tumor heterogeneity observed in glioma of the central nervous tissues.

In this video, we describe a simple and efficient protocol using gradient centrifugation to isolate single nuclei. Once isolated, these nuclei can be used for sequencing and genome analysis.

This video demonstrates the cryopreservation of a mouse’s brain to isolate discrete anatomical areas or specific cell populations from the tumor tissues. This technique is critical in obtaining optimal optical resolution of brain tissue morphology for subsequent analysis.

In this video, we demonstrate the sectioning of a frozen mouse brain tumor tissue using a cryostat. The frozen brain tissue sections so obtained are stored at low temperatures until further analysis.

This video presents the fixation and staining method to process frozen mouse brain tumor tissue. The processed tissue helps differentiate and obtain a specific population of cells during subsequent laser capture microdissection.

This video describes a method to extract and purify ubiquitinylated peptides containing remnant di-glycine peptides from a complex peptide mixture. The presented method may help in identifying original ubiquitination sites in the protein.

This video describes a method based on size-exclusion chromatography (SEC) to isolate extracellular vesicles or EVs derived from the brain immune cells―the microglia. The EVs act as molecular cargo to deliver mediators, which regulate an immune response. Therefore, the isolated EVs can be used as an in vitro model to study the microglia-neuron crosstalk during a neuronal immune response.

In this video, we demonstrate the SDS-PAGE in-gel digestion method for protein extraction from extracellular vesicles or EVs. Once isolated, the obtained protein fragments can be used for proteomic analysis.

In this video, we study tumor-macrophage interaction using fluorescence-based transwell macrophage interaction assay. The assay involves the analysis of the migration of macrophages through chemical-induced chemotaxis.

In this video, we determine the proliferative capacity of neuronal progenitor cells by measuring the incorporation of radioactive tracer tritiated thymidine into the DNA of cells. This technique can be used to study cell proliferation after treatment with certain drugs and growth factors.

This video demonstrates the neural precursor cell neurosphere migration assay—an in vitro assay to study the migratory response of neural precursor cells from the neurosphere—via microscopic analysis.

This video describes the technique of extracting extracellular vesicles from fresh or frozen tissues. The isolated extracellular vesicles can be used for further analysis to uncover their significant roles in disease pathogenesis.

This video demonstrates the density gradient ultracentrifugation-based purification method to isolate subpopulations of extracellular vesicles suitable for characterization studies.

In this video, we describe the method to obtain a pure preparation of astrocytes from newly born mice.

This video presents the isolation of microvessels from a rat brain using a combination of density gradient centrifugation and enzymatic digestion mehods. The isolated microvessels can be cultured in a culture flask pre-coated with collagen and fibronectin matrix to obtain endothelial cell colonies.

In this video, we describe a comprehensive protocol to establish an in vitro model mimicking the blood-brain tumor barrier. The model holds promise to understand the delivery of therapeutic agents for the treatment of brain tumors.

This video describes a permeability assay to evaluate the permeability of in vitro blood-brain barrier using sodium fluorescein molecules.

In this video, we isolate zebrafish microglial cells via immunostaining-coupled cell sorting. The isolated microglia can be used in downstream experiments to understand their role in brain pathologies.

This video demonstrates the reprogramming of peripheral blood mononuclear cells (PBMNCs) into induced neural stem cells (iNSCs) using Sendai virus. Sendai virus is a non-integrating virus that offers an efficient and safe vehicle for reprogramming cells.

In this video, we demonstrate the repetitive tumor challenge assay to evaluate the CAR T cells’ repetitive tumor killing potential during high tumor cell loads. Further, the CAR T cells can be analyzed using flow cytometry to determine the different subpopulations of CAR T cells in the sample.

This video describes a silica-based membrane procedure to isolate circulating cell-free DNA or cfDNA from human plasma sample. The cfDNA is a promising biomarker for cancer diagnosis and...

In this video, we analyze the size distribution of cell-free DNA or cfDNA fragments using automated capillary electrophoresis. This technique helps gain insights into the profile of DNA fragments released by cancer cells during apoptosis.

In this video, we present a step-by-step procedure to generate emulsified nano-droplets to detect target nucleic acids by digital polymerase chain reaction or dPCR.

This video describes the technique of seeding mesenchymal stem cells (MSCs) onto a biodegradable poly(lactic acid) (PLA) scaffold. The prepared scaffolds can be implanted into the surgical resection cavity of the brain from where they may migrate towards tumor foci. Further, these cells can be engineered to express therapeutic proteins to stimulate apoptosis in cancer cells.

In this video, we describe a protocol for genetically engineering glioma cells to express infrared fluorescent protein or iRFP. These fluorescent-tagged glioma cells hold utility in deep tissue imaging to understand the development of brain tumors.

In this video, we perform brain tumor stem cell migration assay to analyze the effect of the drug of interest on the migration of live brain tumor stem cells using chemotaxis migration plate. Live-cell imaging facilitates the visualization and quantification of cell migration during the assay.

This video demonstrates the in vitro brain tumor stem cell (BTSC) invasion assay to determine the effect of drug treatment on the invasion capacity of BTSCs from the neurosphere via live-cell imaging technique.

This video describes a co-culture assay of glioblastoma cells on micropatterned neurons, which mimic the myelinated neuronal tracts invasion by the glioblastoma stem-like cells as they migrate on neurons in the peritumoral spaces. This assay can help analyze the effects of pharmacological drugs that inhibit glioblastoma cells' migration on neurons in a glioma.

In this video, we demonstrate the procedure to isolate and culture pericytes from bovine brain capillaries without endothelial cell contamination.

In this video, we perform zymography, an electrophoretic technique, to analyze the proteolytic activity within biological samples.

In this video, we generate microtumors from patient-derived tumor cells using 3D human biogel. These microtumors closely resemble the in vivo tumor microenvironment and serve as potential platforms for drug testing.

In this video, we evaluate the phagocytic capacity of purified astrocytes in vitro by live imaging assay. The assay serves as an effective screening platform to identify chemical compounds that can enhance or inhibit the phagocytic capacity of astrocytes.

In this video, we perform electroporation of chick embryo cerebellum slices with reporter gene plasmids. The technique facilitates visualization of the development and migration of granule cell precursors to form granular layer.

This video demonstrates the protocol for single-cell electroporation of genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice cultures. The technique is useful to examine specific molecular and physiological functions of neurons, including cell autonomous mechanisms and transsynaptic protein interactions.

In this video, we describe the surgical procedure to create a calvarial window for accessing the brain of a mouse model.

This video demonstrates implantation of tumor cells in the mouse brain by performing intracranial injection.

In this video, we describe the procedure to deliver human-derived tumor cells into the cisterna magna of a mouse’s brain. The orthotopic mouse model can be used to study metastatic spread of cancer to the cranial meninges.

This video demonstrates ommaya mediated intracerebroventricular injection in a mouse model. The method enables localized delivery of potential drug candidates and evades the blood-brain barrier.

This video presents a surgical procedure to implant cranial window in a mouse model for stable optical access to different regions of the brain.

In this video, we perform intravital imaging of pre-injected cancer cells in a mouse model via an implanted cranial window. This high-resolution time-lapse imaging technique enables the study of dynamic behavior of cancer cells in the live mouse.

This video demonstrates the application of Tumor Treating Fields or TTFields in human brain tumor models. This non-invasive technique delivers low-intensity alternating electric fields directly to the brain, resulting in inhibition of cell division and subsequent apoptosis.

This video presents the surgical procedure for image-guided tumor resection and implantation of a bio-scaffold seeded with genetically engineered mesenchymal stem cells or MSCs expressing tumor necrosis factor-related apoptosis-inducing ligand or TRAIL. These therapeutic MSCs have the potential in treatment of brain tumors.

In this video, we describe fluorescence molecular tomography or FMT for in vivo imaging of fluorescent protein-tagged glioblastoma xenografts in mouse models. The technique is useful for elucidating the effects of anti-tumor therapeutics at the molecular level.

This video describes a method of temporarily disrupting the blood-brain barrier (BBB) by creating transient openings using microbubble-mediated focused ultrasound (FUS). These transient local BBB openings can eventually be used to deliver larger-sized drug molecules directly into the brain parenchyma.

This video demonstrates the delivery of plasmid DNA into cerebellar neuronal cells of murine embryos using in utero electroporation. This method is useful to gain understanding of in vivo function of genes in normal brain development and the changes in gene expression of neuron cells during tumor progression.

In this video, we develop an in vivo mouse model for perineural invasion by injecting pancreatic cancer cells into the sciatic nerve. This model helps quantify the extent of invasion and study the interactions between cancer cells and nerves.

This video demonstrates the establishment of a brain metastasis mouse model by intracarotid injection of fluorescently labeled cancer cells. The mouse model can be used to study the effects of novel therapeutics and the underlying physiological mechanisms.

This video demonstrates the orthotopic transplantation of zebrafish brain tumor cells into the fourth ventricle of an immunocompetent zebrafish embryo. This procedure allows for the long-term study of tumor cell invasion and behavior.

This video demonstrates the intranasal delivery of oncolytic virus loaded therapeutic neural stem cells into a brain-tumor bearing mouse model. The non-invasive intranasal administration of stem cells bypasses the blood brain barrier, facilitating their delivery to the tumor site in the brain for therapeutic applications.

This video describes an efficient technique to induce glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate, and generate tumors with histopathological characteristics of human GBM.

This video demonstrates the isolation of intact vessels from a mouse's brain that constitute the blood-brain barrier (BBB) along with neurons and glial cells. The isolated vessels can be used as an in vitro model to study their role in maintaining brain homeostasis.

In this video, we perform the isolation of mononuclear cells from a mouse’s brain using density gradient centrifugation. The isolated mononuclear cell suspension can be used for further downstream analysis.

This video describes a protocol to isolate neurons, macrophages, and microglia from larval zebrafish brains by mechanically disrupting the brain tissue. The purified cell populations can further be used for genomic analysis.

This video presents the isolation of cerebral capillaries from a fresh human brain tissue. The isolated capillaries can then be used to generate an ex vivo blood brain barrier model.

In this video, we describe the procedure to establish a murine model of perineural invasion by injecting cancer cells into the sciatic nerve. The model can help gain an understanding of the magnitude of nerve invasion for subsequent assessment of anti-cancer drugs.

This video describes a method to generate tissue slices from the mouse cerebellum. The cerebellar slices can be used for organotypic culturing that provides insights into neurodevelopmental mechanisms, including myelination dynamics.

Source: Eldeiry, M. et al. Spinal Cord Neurons Isolation and Culture from Neonatal Mice. J. Vis. Exp. (2017)In this video, we describe a step-by-step protocol for isolating spinal cord neurons from a neonatal mouse for in vitro studies.