1Molecular Pharmacology and Pathology Program, Department of Pathology & Bosch Institute, University of Sydney, 2Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University
Ascorbate plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. Here we describe a medium-throughput, specific and inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.
Published April 11, 2014. Keywords: Biochemistry, Vitamin C, Ascorbate, Cell swelling, Glutamate, Microplate assay, Astrocytes
1Department of Experimental Oncology, European Institute of Oncology
By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.
Published April 11, 2014. Keywords: Biochemistry, chromatin, histone post-translational modifications (hPTMs), epigenetics, mass spectrometry, proteomics, SILAC, chromatin immunoprecipitation, histone variants, chromatome, hPTMs cross-talks
1Department of Biomedical Sciences, Dr. William M Scholl College of Podiatric Medicine, Rosalind Franklin University of Medicine and Science, 2Department of Cell Biology and Anatomy, Rosalind Franklin University of Medicine and Science
Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.
Published April 8, 2014. Keywords: Developmental Biology, Zebrafish, lateral line regeneration, lateral line development, neuromasts, hair cell regeneration, disease models
1Stem Cell Institute, Paul and Sheila Wellstone Muscular Dystrophy Center, Department of Neurology, University of Minnesota Medical School
The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.
Published April 8, 2014. Keywords: Cellular Biology, skeletal muscle, muscle stem cell, satellite cell, regeneration, myoblast transplantation, muscular dystrophy, self-renewal, differentiation, myogenesis
1Center for Vascular Biology Research, Department of of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School
Nanopodia are thin but fragile membrane channels that extend up to 100 μm from a cell's leading front or trailing rear and sense the cellular environment. Direct fixation at 37 °C, gentle washing, and avoidance of organic solvents like ethanol, methanol, or acetone and of higher Triton X-100 concentrations are required to observe these cellular structures.
Published April 3, 2014. Keywords: Cellular Biology, nanopodia, TM4SF1, endothelial cell, tumor cell, F-actin, immunofluorescence staining, tetraspanin
1Department of Biological Sciences, Wayne State University
Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.
Published April 2, 2014. Keywords: Biochemistry, Chromaffin Cells, Lipid Bilayers, Microscopy, Fluorescence, Polarization, Exocytosis, membrane, TIRF, pTIRF, chromaffin, polarization, vesicle
1Institute of Laboratory Animal Science, University of Zurich, 2Department of Genetics, Cell Biology & Development and Center for Genome Engineering, University of Minnesota
Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.
Published April 2, 2014. Keywords: Genetics, Oocyte microinjection, Designer nucleases, ZFN, TALEN, Genome Engineering
1Department of Biochemistry and Cell Biology, Stony Brook University, State University of New York
Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular “tags” for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.
Published March 26, 2014. Keywords: Biochemistry, Ubiquitin/proteasome system, 26S proteasome, protein degradation, proteasome inhibitor, Western blotting, plant genetic transformation
1Biological Sciences in Dental Medicine, Harvard University, 2Biology Department, Massachusetts Institute of Technology, 3Whitehead Institute, Massachusetts Institute of Technology, 4Biology Department, Virginia Commonwealth University
A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.
Published March 26, 2014. Keywords: Developmental Biology, craniofacial development, neural crest, Mouth, Nostril, transplantation, Xenopus
1Department of Biochemistry, Purdue University
Drosophila tissues often contain a heterogeneous mixture of cell types. To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.
Published March 25, 2014. Keywords: Biochemistry, Gene Expression, nuclei isolation, Drosophila, KASH, GFP, cell-type specific