1GIBCO, Life Technologies
This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.
Published July 15, 2010. Keywords: Cellular Biology, iPS, pluripotent, stem cells, cell culture, medium, media, feeder-free
1Department of Molecular Physiology and Biophysics, Baylor College of Medicine, 2Department of Neuroscience, Baylor College of Medicine, 3Department of Neuroscience, University of California at San Diego, 4National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine
To preserve neuronal processes for ultrastructural analysis, we describe a protocol for plating of primary neurons on electron microscopy grids followed by flash freezing, yielding samples suspended in a layer of vitreous ice. These samples can be examined with a cryo-electron microscope to visualize structures at the nanometer scale.
Published February 12, 2014. Keywords: Neuroscience, Neurons, Cryo-electron Microscopy, Electron Microscope Tomography, Brain, rat, primary neuron culture, morphological assay
1School of Biosciences, University of Birmingham
The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.
Published September 21, 2010. Keywords: Cellular Biology, Skeletal muscle stem cell, embryonic tissue culture, apoptosis, growth factor, proliferation, myoblast, myogenesis, satellite cell, skeletal muscle differentiation, muscular dystrophy
1Department of Neurobiology and Behavior, University of California, Irvine
Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.
Published November 11, 2010. Keywords: Neuroscience, cryopreservation, neuron, astrocyte, neuronal stem cells, tissue culture, brain cortex
JoVE Clinical and Translational Medicine
1Department of Neurosurgery, Henry Ford Hospital
Protocol for propagation of dissociated high grade glioma surgical specimens in serum-free neurosphere medium to select for cells with cancer stem cell phenotype. For specimens that fail to grow as neurospheres, an alternative protocol is suggested. A paraffin embedding technique for maintaining the 3D neurosphere architecture for immunocytochemistry is described.
Published January 7, 2014. Keywords: Medicine, Primary Cell Culture, animal models, Nervous System Diseases, Neoplasms, glioblastoma, neurosphere, surgical specimens, long-term self-renewal
1Centre d’Immunologie de Marseille-Luminy, Université de la Méditerranée
We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.
Published February 27, 2012. Keywords: Molecular Biology, C. elegans, fluorescent reporter, Biosort, LIMS, innate immunity, Drechmeria coniospora
1Department of Biology, University of North Carolina at Chapel Hill
Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.
Published October 31, 2013. Keywords: Microbiology, High-Throughput Screening Assays, Genes, Reporter, Microbial Interactions, Soil Microbiology, Coculture, microbial interactions, screen, fluorescent transcriptional reporters, Bacillus subtilis
1Randall and Cardiovascular Divisions, King’s College London, 2Division of Cardiology, School of Medicine, University of California San Diego
Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.
Published September 6, 2013. Keywords: Cellular Biology, Biomedical Engineering, Bioengineering, Molecular Biology, Cell Culture Techniques, Primary Cell Culture, Cell Culture Techniques, Primary Cell Culture, Cell Culture Techniques, Primary Cell Culture, Cell Culture Techniques, Disease Models, Animal, Models, Cardiovascular, Cell Biology, neonatal mouse, cardiomyocytes, isolation, culture, primary cells, NMC, heart cells, animal model
1Animal Science Department, Iowa State University
Preimplantation embryos may be cryopreserved after placement into a hypertonic cryoprotective solution to cause cellular dehydration. After equilibration, ice crystal formation is induced in the solution surrounding the embryo. Further dehydration occurs as the embryo is slowly cooled to subzero temperatures before plunging into liquid nitrogen for storage.
Published August 5, 2011. Keywords: Developmental Biology, embryo, cryopreservation, cattle, sheep, goats
1Research and Development, Stemgent
Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.
Published December 24, 2009. Keywords: Developmental Biology, Human embryonic stem cell, medium, hES, maintenance, thaw, ES cells, stem cell, cell culture, pluripotency, differentiation, passage, freeze