NS21: re-defined and modified supplement B27 for neuronal cultures.

Department of Pharmacology, University of Iowa, Iowa City, IA 52242-1109, United States.
Journal of Neuroscience Methods (Impact Factor: 1.96). 07/2008; 171(2):239-47. DOI: 10.1016/j.jneumeth.2008.03.013
Source: PubMed

ABSTRACT In vitro culturing of primary neurons is a mainstay of neurobiological research. Many of these culture paradigms have taken advantage of defined culture media rather than serum additives that contain undefined survival factors to facilitate experimental manipulations and interpretation of the results. To culture neurons in the absence of serum, defined supplements such as B27 are now widely used. However, commercially available supplements exhibit large variability in their capabilities to support neurons in culture. We re-optimized and modified earlier published formulations of B27 using 21 different ingredients (NS21). NS21 supports neuronal cultures of high quality as manifested by their morphological characteristics, formation of synapses, and postsynaptic responses. Much of the variability in the quality of B27/NS21 was due to variability in the quality of different sources of bovine serum albumin. Furthermore, we found that holo-transferrin used in NS21 is preferable over apo-transferrin used in B27 for the quality of neuronal cultures.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Aims: Determine the mechanism by which CHIP induction alters neuronal survival under conditions of mitochondrial stress induced by oxygen and glucose deprivation. Results: We report that animals deficient in the E3 ubiquitin ligase, CHIP, have high baseline levels of CNS protein oxidation and lipid peroxidation, reduced antioxidant defenses and decreased energetic status. Stress-associated molecules typically linked to Parkinson's disease such as the mitochondrial kinase PINK1 and another E3 ligase, Parkin, are upregulated in brains from CHIP KO animals. Utilizing a novel biotin-avidin capture technique, we found that the oxidation status of Parkin and the mitochondrial fission protein, Drp1 are altered in a CHIP-dependent manner. We also found that following oxygen glucose deprivation (OGD) the expression of CHIP, PINK1 and the autophagic marker LC3 are increased and there is activation of the redox sensitive kinase p66shc. Under conditions of OGD, CHIP relocalizes from the cytosol to mitochondria. Mitochondria from CHIP KO mice have profound impairments in stress response induced by calcium overload resulting in accelerated permeability transition activity. While CHIP deficient neurons are morphologically intact, they are more susceptible to OGD consistent with a previously unknown neuroprotective role for CHIP in maintaining mitochondrial homeostasis. Innovation: CHIP relocalization to the mitochondria is essential for the regulation of mitochondrial integrity and neuronal survival following OGD. Conclusions: CHIP is an essential regulator of neuronal bioenergetics and redox tone. Altering the expression of this protein has profound effects on neuronal survival when cells are exposed to OGD.
    Antioxidants & Redox Signaling 01/2015; [Epub ahead of print]. · 8.20 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Multipotent neural stem cells (NSCs) are currently under investigation as a candidate treatment for central nervous system (CNS) injury because of their potential to compensate for neuronal damage and to reconstruct disrupted neuronal connections. To maximize the regenerative effect of the derived neurons and to minimize the side effects of the derived astrocytes, it is necessary to regulate the fate determination of NSCs to produce more neurons and fewer astrocytes. Both valproic acid (VPA) and all-trans-retinoic acid (ATRA), two clinically established drugs, induce neuronal differentiation and facilitate neurite outgrowth at the expense of astrocytic differentiation in NSCs. However, the time-dependent activities and the long-term treatment effects of these drugs have not been explored in NSCs. More importantly, the efficacies of VPA and ATRA in neuronal promotion and astrocytic suppression remain unclear. In this study, we compare the time-dependent characteristics of VPA and ATRA in NSC differentiation and neurite outgrowth in vitro and, for the first time, demonstrate the improved efficacy of their combined application in neuronal induction and astrocytic suppression. These significant effects are closely coupled to the altered expression of a neurogenic transcription factor, a Wnt signaling component, a cell cycle regulator and a neural growth factor, indicating an underlying cross-talk between the mechanisms of action of ATRA and VPA. These findings indicate that a novel strategy combining these two therapeutic drugs may improve the restorative effect of NSC transplantation by altering the expression of their interconnected targets for fate determination. Copyright © 2014. Published by Elsevier B.V.
    Brain Research 11/2014; DOI:10.1016/j.brainres.2014.11.029 · 2.83 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The role of intermediate methylation states in DNA is unclear. Here, to comprehensively identify regions of intermediate methylation and their quantitative relationship with gene activity, we apply integrative and comparative epigenomics to 25 human primary cell and tissue samples. We report 18,452 intermediate methylation regions located near 36% of genes and enriched at enhancers, exons and DNase I hypersensitivity sites. Intermediate methylation regions average 57% methylation, are predominantly allele-independent and are conserved across individuals and between mouse and human, suggesting a conserved function. These regions have an intermediate level of active chromatin marks and their associated genes have intermediate transcriptional activity. Exonic intermediate methylation correlates with exon inclusion at a level between that of fully methylated and unmethylated exons, highlighting gene context-dependent functions. We conclude that intermediate DNA methylation is a conserved signature of gene regulation and exon usage.
    Nature Communications 02/2015; 6:6363. DOI:10.1038/ncomms7363 · 10.74 Impact Factor

Full-text (2 Sources)

Available from
May 19, 2014

Yucui Chen