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  • Ahlenius, Henrik, et al. (författare)
  • Isolation and generation of neurosphere cultures from embryonic and adult mouse brain.
  • 2010
  • Ingår i: Methods in Molecular Biology. - : Springer. - 1940-6029. ; 633, s. 241-252
  • Tidskriftsartikel (refereegranskat)abstract
    • Neural stem cells are defined as cells that either gives rise to or derives from the cells of the central nervous system and have the unique properties of stem cells, i.e. self-renewal and multipotentiality. One of the widely used methods of expanding neural stem cells under culture conditions is based on the capacity of these cells to divide continuously when cultured in serum-free medium supplemented with various growth factors. One common method used is to grow neural stem cells as free-floating aggregates of cells called neurospheres. Neurospheres can be generated from several structures of the embryonic and adult mammalian brain. Although viable lines can be generated from crude extracts of brain, a precise dissection is crucial to get a pure population of cells. Here we describe methods for dissection, isolation and generation of neurospheres from embryonic ganglionic eminences and adult subventricular zone of mice and rats.
  • Ahmad, Faiyaz, et al. (författare)
  • Adenovirus-mediated overexpression of murine cyclic nucleotide phosphodiesterase 3B
  • 2005
  • Ingår i: Methods in molecular biology (Clifton, N.J.). - : Humana Press. - 1064-3745 .- 1940-6029. ; 307, s. 93-107
  • Bokkapitel (övrigt vetenskapligt)abstract
    • To construct the recombinant adenovirus vector containing the cDNA for recombinant mouse cyclic nucleotide phosphodiesterase 3B (mPDE3B), the cDNA for mPDE3B was subcloned into pACCMV.pLpA. Subsequently, this recombinant plasmid, pACCMV.mPDE3B, was cotransfected with pJM17 plasmid containing the adenoviral genome into 293 human embryonic kidney cells, and the replication-deficient adenovirus AdCMV.mPDE3B was generated via homologous recombination. Large-scale preparation of adenovirus yielded 10(11)-10(13) viral particles/mL and could be quantitated by real-time polymerase chain reaction using iCycler (Bio-Rad). Efficiency of gene transfer was assessed by infecting FDCP2 or H4IIE cells with a recombinant adenovirus expressing beta-galactosidase (beta-gal); greater than 75% of cells were infected. Expression of mPDE3B in H4IIE hepatoma cells, FDCP2 hematopoietic cells, and beta-cells from isolated pancreatic islets was detected by Western blot analysis. In lysates from FDCP2 cells and H4IIE hepatoma cells infected with recombinant adenoviral mPDE3B constructs, mPDE3B activity was increased 10- to 30-fold compared with the activity in lysates from cells infected with beta-gal adenovirus. Stimulation of FDCP2 cells infected with mPDE3B adenovirus with insulin (100 nM, 10 min) resulted in an approx 1.7-fold increase in endogenous PDE3B and recombinant wild-type PDE3B activities. Infection of rat pancreatic islets resulted in a 5- to 10-fold increase in PDE3B expression and activity and subsequent blunting of insulin secretion. Thus, adenovirus-mediated gene transfer is effective for studying expression and regulation of recombinant PDE3 in insulin-responsive cells as well as insulin-secreting cells.
  • Aldrin-Kirk, Patrick, et al. (författare)
  • Practical Considerations for the Use of DREADD and Other Chemogenetic Receptors to Regulate Neuronal Activity in the Mammalian Brain
  • Ingår i: Methods in molecular biology (Clifton, N.J.). - : Springer. - 1940-6029. ; 1937, s. 59-87
  • Tidskriftsartikel (refereegranskat)abstract
    • Chemogenetics is the process of genetically expressing a macromolecule receptor capable of modulating the activity of the cell in response to selective chemical ligand. This chapter will cover the chemogenetic technologies that are available to date, focusing on the commonly available engineered or otherwise modified ligand-gated ion channels and G-protein-coupled receptors in the context of neuromodulation. First, we will give a brief overview of each chemogenetic approach as well as in vitro/in vivo applications, then we will list their strengths and weaknesses. Finally, we will provide tips for ligand application in each case.Each technology has specific limitations that make them more or less suitable for different applications in neuroscience although we will focus mainly on the most commonly used and versatile family named designer receptors exclusively activated by designer drugs or DREADDs. We here describe the most common cases where these can be implemented and provide tips on how and where these technologies can be applied in the field of neuroscience.
  • Alexandersson, Erik, et al. (författare)
  • Purification and proteomic analysis of plant plasma membranes.
  • 2008
  • Ingår i: Methods in Molecular Biology. - : Springer. - 1940-6029. ; 432, s. 161-173
  • Tidskriftsartikel (refereegranskat)abstract
    • All techniques needed for proteomic analyses of plant plasma membranes are described in detail, from isolation of plasma membranes to protein identification by mass spectrometry (MS). Plasma membranes are isolated by aqueous two-phase partitioning yielding vesicles with a cytoplasmic side-in orientation and a purity of about 95%. These vesicles are turned inside-out by treatment with Brij 58, which removes soluble contaminating proteins enclosed in the vesicles as well as loosely attached proteins. The final plasma membrane preparation thus retains all integral proteins and many peripheral proteins. Proteins are separated by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein bands are excised and digested with trypsin. Peptides in tryptic digests are separated by nanoflow liquid chromatography and either fed directly into an ESI-MS or spotted onto matrix-assisted laser desorption ionization (MALDI) plates for analysis with MALDI-MS. Finally, data processing and database searching are used for protein identification to define a plasma membrane proteome.
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