Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium
This protocol describes the isolation, enrichment, and maintenance of medulloblastoma tumor stem cells derived from mutant mice with ectopic Sonic hedgehog pathway activity.
Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.
Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface
Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium. A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin.
Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique
Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.
1Department of Cell and Developmental Biology, State University of New York, Upstate Medical University, 2Department of Neurobiology and Anatomy, Eccles Institute of Human Genetics, University of Utah
Cilia-generated fluid flow in Kupffer’s Vesicle (KV) controls left-right patterning of the zebrafish embryo. Here, we describe a technique to modulate gene function specifically in KV cells. In addition, we show how to deliver fluorescent beads into KV to visualize fluid flow.
Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.
Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease
Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.
Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording
The electroolfactogram (EOG) recording is an informative, easy-to-conduct, and reliable way of assessing olfactory function at the level of the olfactory epithelium. This protocol describes a recording setup, mouse tissue preparation, data collection, and basic data analysis.
Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.
1Department of Medical Oncology, Dana-Farber Cancer Institute, 2Harvard Medical School, Boston, MA, 3Sheba Cancer Research Center, Chaim Sheba Medical Center, 4Department of Pathology, Brigham and Women's Hospital
The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.
1Department of Otology and Laryngology, Harvard Medical School, 2Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, 3Department of Communication Sciences and Disorders, Emerson College, 4Program in Speech and Hearing Bioscience and Technology, Division of Health Science and Technology, Harvard
This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.
1Department of Neurosurgery, The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco - UCSF, 2Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, 3Department of Neuroscience and Neurology, College of Physicians and Surgeons, Columbia University, 4Department of Developmental and Regenerative Biology, Nagoya City University Graduate School of Medical Sciences, 5Center for Motor Neuron Biology and Disease, College of Physicians and Surgeons, Columbia University
The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region.
Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro
The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.
Procedure for fertilizing Xenopus oocytes by the host transfer method.
We present a method to collect cerebrospinal fluid (CSF) and to create a system which lacks CSF within the embryonic zebrafish brain ventricular system. This allows for further examination of CSF composition and its requirement during embryonic brain development.
A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.
We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.
Application and direct measurements of forces on neurons in the 2-1000 microdyne range are achieved with high precision using calibrated glass needles. This methodology can be used to control and measure several aspects of axonal development, including axonal initiation, axonal tension, velocity of axonal elongation, and force vectors.
1Institute of Pathology, Laboratory of Molecular Tumor Pathology, Charité - Universitätsmedizin Berlin, 2Institute for Chemistry and Biochemistry, Free University Berlin, 3Laboratory for Functional Genomics Charité (LFGC), Charité - Universitätsmedizin Berlin, 4Comprehensive Cancer Center Charité, Charité - Universitätsmedizin Berlin
This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).