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  • Consistent injury to medium spiny neurons and white matter in the mouse striatum after prolonged transient global cerebral ischemia. 21309724

    A reproducible transient global cerebral ischemia (tGCI) mouse model has not been fully established. Although striatal neurons and white matter are recognized to be vulnerable to ischemia, their injury after tGCI in mice has not been elucidated. The purpose of this study was to evaluate injuries to striatal neurons and white matter after tGCI in C57BL/6 mice, and to develop a reproducible tGCI model. Male C57BL/6 mice were subjected to tGCI by bilateral common carotid artery occlusion (BCCAO). Mice whose cortical cerebral blood flow after BCCAO decreased to less than 13% of the pre-ischemic value were used. Histological analysis showed that at 3 days after 22 min of BCCAO, striatal neurons were injured more consistently than those in other brain regions. Quantitative analysis of cytochrome c release into the cytosol and DNA fragmentation in the striatum showed consistent injury to the striatum. Immunohistochemistry and Western blot analysis revealed that DARPP-32-positive medium spiny neurons, the majority of striatal neurons, were the most vulnerable among the striatal neuronal subpopulations. The striatum (especially medium spiny neurons) was susceptible to oxidative stress after tGCI, which is probably one of the mechanisms of vulnerability. SMI-32 immunostaining showed that white matter in the striatum was also consistently injured 3 days after 22 min of BCCAO. We thus suggest that this is a tGCI model using C57BL/6 mice that consistently produces neuronal and white matter injury in the striatum by a simple technique. This model can be highly applicable for elucidating molecular mechanisms in the brain after global ischemia.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • KSR-based medium improves the generation of high-quality mouse iPS cells. 25171101

    Induced pluripotent stem (iPS) cells from somatic cells have great potential for regenerative medicine. The efficiency in generation of iPS cells has been significantly improved in recent years. However, the generation of high-quality iPS cells remains of high interest. Consistently, we demonstrate that knockout serum replacement (KSR)-based medium accelerates iPS cell induction and improves the quality of iPS cells, as confirmed by generation of chimeras and all iPS cell-derived offspring with germline transmission competency. Both alkaline phosphatase (AP) activity assay and expression of Nanog have been used to evaluate the efficiency of iPS cell induction and formation of ES/iPS cell colonies; however, appropriate expression of Nanog frequently indicates the quality of ES/iPS cells. Interestingly, whereas foetal bovine serum (FBS)-based media increase iPS cell colony formation, as revealed by AP activity, KSR-based media increase the frequency of iPS cell colony formation with Nanog expression. Furthermore, inhibition of MAPK/ERK by a specific inhibitor, PD0325901, in KSR- but not in FBS-based media significantly increases Nanog-GFP+ iPS cells. In contrast, addition of bFGF in KSR-based media decreases proportion of Nanog-GFP+ iPS cells. Remarkably, PD can rescue Nanog-GFP+ deficiency caused by bFGF. These data suggest that MAPK/ERK pathway influences high quality mouse iPS cells and that KSR- and PD-based media could enrich homogeneous authentic pluripotent stem cells.
    Document Type:
    Reference
    Product Catalog Number:
    MAB4301
    Product Catalog Name:
    Anti-Stage-Specific Embryonic Antigen-1 Antibody, clone MC-480

  • Optimizing the medium perfusion rate in bone tissue engineering bioreactors. 21449028

    There is a critical need to increase the size of bone grafts that can be cultured in vitro for use in regenerative medicine. Perfusion bioreactors have been used to improve the nutrient and gas transfer capabilities and reduce the size limitations inherent to static culture, as well as to modulate cellular responses by hydrodynamic shear. Our aim was to understand the effects of medium flow velocity on cellular phenotype and the formation of bone-like tissues in three-dimensional engineered constructs. We utilized custom-designed perfusion bioreactors to culture bone constructs for 5 weeks using a wide range of superficial flow velocities (80, 400, 800, 1,200, and 1,800 µm/s), corresponding to estimated initial shear stresses ranging from 0.6 to 20 mPa. Increasing the flow velocity significantly affected cell morphology, cell-cell interactions, matrix production and composition, and the expression of osteogenic genes. Within the range studied, the flow velocities ranging from 400 to 800 µm/s yielded the best overall osteogenic responses. Using mathematical models, we determined that even at the lowest flow velocity (80 µm/s) the oxygen provided was sufficient to maintain viability of the cells within the construct. Yet it was clear that this flow velocity did not adequately support the development of bone-like tissue. The complexity of the cellular responses found at different flow velocities underscores the need to use a range of evaluation parameters to determine the quality of engineered bone.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • ΔFosB induction in striatal medium spiny neuron subtypes in response to chronic pharmacological, emotional, and optogenetic stimuli. 24259563

    The transcription factor, ΔFosB, is robustly and persistently induced in striatum by several chronic stimuli, such as drugs of abuse, antipsychotic drugs, natural rewards, and stress. However, very few studies have examined the degree of ΔFosB induction in the two striatal medium spiny neuron (MSN) subtypes. We make use of fluorescent reporter BAC transgenic mice to evaluate induction of ΔFosB in dopamine receptor 1 (D1) enriched and dopamine receptor 2 (D2) enriched MSNs in ventral striatum, nucleus accumbens (NAc) shell and core, and in dorsal striatum (dStr) after chronic exposure to several drugs of abuse including cocaine, ethanol, Δ(9)-tetrahydrocannabinol, and opiates; the antipsychotic drug, haloperidol; juvenile enrichment; sucrose drinking; calorie restriction; the serotonin selective reuptake inhibitor antidepressant, fluoxetine; and social defeat stress. Our findings demonstrate that chronic exposure to many stimuli induces ΔFosB in an MSN-subtype selective pattern across all three striatal regions. To explore the circuit-mediated induction of ΔFosB in striatum, we use optogenetics to enhance activity in limbic brain regions that send synaptic inputs to NAc; these regions include the ventral tegmental area and several glutamatergic afferent regions: medial prefrontal cortex, amygdala, and ventral hippocampus. These optogenetic conditions lead to highly distinct patterns of ΔFosB induction in MSN subtypes in NAc core and shell. Together, these findings establish selective patterns of ΔFosB induction in striatal MSN subtypes in response to chronic stimuli and provide novel insight into the circuit-level mechanisms of ΔFosB induction in striatum.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Expansion of neurofilament medium C terminus increases axonal diameter independent of increases in conduction velocity or myelin thickness. 22553027

    Maturation of the peripheral nervous system requires specification of axonal diameter, which, in turn, has a significant influence on nerve conduction velocity. Radial axonal growth initiates with myelination, and is dependent upon the C terminus of neurofilament medium (NF-M). Molecular phylogenetic analysis in mammals suggested that expanded NF-M C termini correlated with larger-diameter axons. We used gene targeting and computational modeling to test this new hypothesis. Increasing the length of NF-M C terminus in mice increased diameter of motor axons without altering neurofilament subunit stoichiometry. Computational modeling predicted that an expanded NF-M C terminus extended farther from the neurofilament core independent of lysine-serine-proline (KSP) phosphorylation. However, expansion of NF-M C terminus did not affect the distance between adjacent neurofilaments. Increased axonal diameter did not increase conduction velocity, possibly due to a failure to increase myelin thickness by the same proportion. Failure of myelin to compensate for larger axonal diameters suggested a lack of plasticity during the processes of myelination and radial axonal growth.
    Document Type:
    Reference
    Product Catalog Number:
    CMTI-1
    Product Catalog Name:
    EmbryoMax® Embryonic Stem Cell Line - Strain 129/SVEV, passage 11