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: 2.05). 07/2008; 171(2):239-47. DOI: 10.1016/j.jneumeth.2008.03.013
Source: PubMed


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.

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    • "Some critical experiments of the distribution of synaptic proteins were analyzed using a blinding test to minimise the effects of biases. As dendritic spine formation is highly sensitive to culture conditions, such as the quality of B27 supplement (Chen et al., 2008), each set of experiments was usually repeated within a month using the same lot of B27 supplement and culture medium to reduce the variation. "
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    ABSTRACT: Dendritic spines are the major locations of excitatory synapses in the mammalian central nervous system. The transformation from dendritic filopodia to dendritic spines has been recognized as one type of spinogenesis. For instance, syndecan-2 (SDC2), a synaptic heparan sulfate proteoglycan, is highly concentrated at dendritic spines and required for spinogenesis. It induces dendritic filopodia formation, followed by spine formation. However, the molecular regulation of the filopodium-spine transition induced by SDC2 is still unclear. In this report, we show that calcium is an important signal downstream of SDC2 in regulation of filopodium-spine transition but not filopodia formation. SDC2 interacted with the postsynaptic proteins CASK and LIN7 and further recruited NMDAR to the tips of filopodia induced by SDC2. Calcium influx via NMDAR promoted spine maturation because addition of EDTA or AP5 to the culture medium effectively prevented morphological change from dendritic filopodia to dendritic spines. Our data also indicated that F-actin rearrangement regulated by calcium influx is involved in the morphological change, because the knockdown of gelsolin, a calcium-activated F-actin severing molecule, impaired the filopodium-spine transition induced by SDC2. In conclusion, our study demonstrates that postsynaptic proteins coordinate to trigger calcium signalling and cytoskeleton rearrangement and consequently control filopodium-spine transition. © 2014 Wiley Periodicals, Inc. Develop Neurobiol, 2014
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    • "Primary hippocampal neuronal cultures were prepared as previously described [18]. In brief, timed pregnant rats Sprague-Dawley rats (Charles River Laboratories International, Wilmington, MA, USA) were anesthetized with isoflurane. "
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    Full-text · Article · Sep 2014 · PLoS ONE
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    • "and include an array of amino acids and vitamins as well as traditional salts. Gem21 was used as previously described (Chen et al., 2008), and provided insulin, T3, and the antioxidant agents noted above. (Gemini Bio-Products Material Data Safety Sheet; www. "
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    ABSTRACT: Microglia play an important role in fine-tuning neuronal activity. In part, this involves their production of tumor necrosis factor-alpha (TNFα), which increases neuronal excitability. Excessive synaptic activity is necessary to initiate spreading depression (SD). Increased microglial production of proinflammatory cytokines promotes initiation of SD, which, when recurrent, may play a role in conversion of episodic to high frequency and chronic migraine. Previous work shows that this potentiation of SD occurs through increased microglial production of TNFα and reactive oxygen species, both of which are associated with an M1-skewed microglial population. Hence, we explored the role of microglia and their M1 polarization in SD initiation. Selective ablation of microglia from rat hippocampal slice cultures confirmed that microglia are essential for initiation of SD. Application of minocycline to dampen M1 signaling led to increased SD threshold. In addition, we found that SD threshold was increased in rats exposed to environmental enrichment. These rats had increased neocortical levels of interleukin-11 (IL-11), which decreases TNFα signaling and polarized microglia to an M2a-dominant phenotype. M2a microglia reduce proinflammatory signaling and increase production of anti-inflammatory cytokines, and therefore may protect against SD. Nasal administration of IL-11 to mimic effects of environmental enrichment likewise increased M2a polarization and increased SD threshold, an effect also seen in vitro. Similarly, application of conditioned medium from M2a polarized primary microglia to slice cultures also increased SD threshold. Thus, microglia and their polarization state play an essential role in SD initiation, and perhaps by extension migraine with aura and migraine. GLIA 2014
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