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  • Endothelial cell responses towards low-fouling surfaces bearing rGD in a three-dimensional environment. 21679704

    This study reveals that it is possible to obtain a specific cell response towards low-fouling carboxymethyl dextran (CMD) surfaces bearing the RGD adhesive peptide in fibrin. To avoid cell sedimentation on surfaces observed in traditional cell culture systems, CMD surfaces bearing RGD were vertically embedded in fibrin containing human umbilical vein endothelial cells (HUVEC) and their effect over cells was investigated. Compared to the CMD surfaces and to CMD layers bearing the negative control RGE, RGD coatings promoted cell adhesion, induced focal contact formation indicated by co-localization of vinculin and actin fibers, and presented a significant effect over HUVEC net growth during the first 24h of the culture, as revealed by Ki67 staining and cell counting. The intracellular localization of caveolin-1 combined with the expression of beta 1 integrins was investigated and the orientation of HUVEC towards and on the RGD surfaces was studied. When compared to the negative controls, HUVEC responded to the RGD surface in fibrin resulting in acceleration of morphological changes. RGD surfaces supported fibrin degradation by HUVEC as revealed by fluorescent fibrin experiments as well as multi-cellular structure formation, vacuolation and lumen formation.Copyright © 2011 Elsevier Inc. All rights reserved.
    Document Type:
    Reference
    Product Catalog Number:
    AB2910
    Product Catalog Name:
    Anti-Mcl-1 Antibody

  • Control of neural cell composition in poly(ethylene glycol) hydrogel culture with soluble factors. 21823990

    Poly(ethylene glycol) (PEG) hydrogels are being developed as cell delivery vehicles that have great potential to improve neuronal replacement therapies. Current research priorities include (1) characterizing neural cell growth within PEG hydrogels relative to standard culture systems and (2) generating neuronal-enriched populations within the PEG hydrogel environment. This study compares the percentage of neural precursor cells (NPCs), neurons, and glia present when dissociated neural cells are seeded within PEG hydrogels relative to standard monolayer culture. Results demonstrate that PEG hydrogels enriched the initial cell population for NPCs, which subsequently gave rise to neurons, then to glia. Relative to monolayer culture, PEG hydrogels maintained an increased percentage of NPCs and a decreased percentage of glia. This neurogenic advantage of PEG hydrogels is accentuated in the presence of basic fibroblast growth factor and epidermal growth factor, which more potently increase NPC and neuronal expression markers when applied to cells cultured within PEG hydrogels. Finally, this work demonstrates that glial differentiation can be selectively eliminated upon supplementation with a γ-secretase inhibitor. Together, this study furthers our understanding of how the PEG hydrogel environment influences neural cell composition and also describes select soluble factors that are useful in generating neuronal-enriched populations within the PEG hydrogel environment.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Functional neural stem cell isolation from brains of adult mutant SOD1 (SOD1(G93A)) transgenic amyotrophic lateral sclerosis (ALS) mice. 20810028

    OBJECTIVES: The aim of present study is to investigate more functional neural stem cells (NSCs) could be isolated from brains with amyotrophic lateral sclerosis (ALS) and expanded in vitro, based on previous reports demonstrating de novo neurogenesis is enhanced to replace degenerating neural tissue.METHODS: Thirteen- or eighteen-week-old mutant human Cu/Zn superoxide dismutase (SOD1(G93A)) transgenic ALS and wild-type SOD1 transgenic control mice were utilized. Changes in numbers of NSCs in the dentate gyrus were analyzed by immunohistochemistry against nestin and CD133. NSCs were primarily cultured from hippocampus of ALS or control mice. Expression of NSC markers, in vitro expansion capacity, and differentiating potential were compared.RESULTS: Hippocampus of 13-week-old pre-symptomatic ALS mice harbor more cells that can be propagated for more than 12 passages in vitro, compared with same age control mice. Primarily-cultured cells formed neurospheres in the NSC culture medium, expressed NSC markers, and differentiated into cells with differentiated neural cell characteristics in the differentiation condition confirming that they are NSCs. In contrast, long-term expansible NSCs could not be derived from brains of 18-week-old symptomatic ALS mice with the same experimental techniques, although they had comparable nestin-immunoreactive cells in the dentate gyrus.DISCUSSION: These results would suggest that increased neuroregeneration in early phase of ALS could be translated to regenerative approaches; however, long-term exposure to ALS microenvironments could abolish functional capacities of NSCs.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • iRFP is a sensitive marker for cell number and tumor growth in high-throughput systems. 24200967

    GFP and luciferase are used extensively as markers both in vitro and in vivo although both have limitations. The utility of GFP fluorescence is restricted by high background signal and poor tissue penetrance. Luciferase throughput is limited in vitro by the requirement for cell lysis, while in vivo, luciferase readout is complicated by the need for substrate injection and the dependence on endogenous ATP. Here we show that near-infrared fluorescent protein in combination with widely available near-infrared scanners overcomes these obstacles and allows for the accurate determination of cell number in vitro and tumor growth in vivo in a high-throughput manner and at negligible per-well costs. This system represents a significant advance in tracking cell proliferation in tissue culture as well as in animals, with widespread applications in cell biology.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1501
    Product Catalog Name:
    Anti-Actin Antibody, clone C4

  • IFN-α inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome. 22251702

    HBV infection remains a leading cause of death worldwide. IFN-α inhibits viral replication in vitro and in vivo, and pegylated IFN-α is a commonly administered treatment for individuals infected with HBV. The HBV genome contains a typical IFN-stimulated response element (ISRE), but the molecular mechanisms by which IFN-α suppresses HBV replication have not been established in relevant experimental systems. Here, we show that IFN-α inhibits HBV replication by decreasing the transcription of pregenomic RNA (pgRNA) and subgenomic RNA from the HBV covalently closed circular DNA (cccDNA) minichromosome, both in cultured cells in which HBV is replicating and in mice whose livers have been repopulated with human hepatocytes and infected with HBV. Administration of IFN-α resulted in cccDNA-bound histone hypoacetylation as well as active recruitment to the cccDNA of transcriptional corepressors. IFN-α treatment also reduced binding of the STAT1 and STAT2 transcription factors to active cccDNA. The inhibitory activity of IFN-α was linked to the IRSE, as IRSE-mutant HBV transcribed less pgRNA and could not be repressed by IFN-α treatment. Our results identify a molecular mechanism whereby IFN-α mediates epigenetic repression of HBV cccDNA transcriptional activity, which may assist in the development of novel effective therapeutics.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
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    Multiple

  • Serum inhibitors for human mast cell growth: possible role of retinol. 14510724

    BACKGROUND: In vitro culture systems have been used to study the physiological and pathological characteristics of human mast cells. However, there are some differences in proliferation and maturation of mast cells between fetal bovine serum (FBS)-containing and serum-deprived cultures. Accordingly, we attempted to identify circulating factor(s) affecting the development of human mast cells. METHODS: We measured the serum levels of retinol and several cytokines. To elucidate the antiproliferative effects of the serum, a retinoic acid receptor (RARalpha) antagonist and neutralizing antibodies against cytokines were used. RESULTS: Similar to FBS, human serum dose-dependently suppressed the growth of tryptase+ cells from CD34+ cord blood cells or 20-week cultured mast cells under stimulation with stem cell factor (SCF). The serum-mediated inhibition might be based on a decline in proliferation rate. Among inhibitors for mast cell growth, retinol and transforming growth factor (TGF)-beta1 were present at high levels in human serum. In contrast with anti-TGF-beta1 antibody, an RARalpha antagonist counteracted the serum-induced suppression of human mast cell proliferation. CONCLUSIONS: Our results suggest that retinol and its derivatives act as a circulating regulator for human mast cell growth. The RARalpha antagonist may be a useful tool to obtain higher numbers of mast cells in FBS-containing cultures.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1222
    Product Catalog Name:
    Anti-Tryptase Antibody, Mast Cell, clone G3

  • Subcellular localization of LGN during mitosis: evidence for its cortical localization in mitotic cell culture systems and its requirement for normal cell cycle progressi ... 12925752

    Mammalian LGN/AGS3 proteins and their Drosophila Pins orthologue are cytoplasmic regulators of G-protein signaling. In Drosophila, Pins localizes to the lateral cortex of polarized epithelial cells and to the apical cortex of neuroblasts where it plays important roles in their asymmetric division. Using overexpression studies in different cell line systems, we demonstrate here that, like Drosophila Pins, LGN can exhibit enriched localization at the cell cortex, depending on the cell cycle and the culture system used. We find that in WISH, PC12, and NRK but not COS cells, LGN is largely directed to the cell cortex during mitosis. Overexpression of truncated protein domains further identified the Galpha-binding C-terminal portion of LGN as a sufficient domain for cortical localization in cell culture. In mitotic COS cells that normally do not exhibit cortical LGN localization, LGN is redirected to the cell cortex upon overexpression of Galpha subunits of heterotrimeric G-proteins. The results also show that the cortical localization of LGN is dependent on microfilaments and that interfering with LGN function in cultured cell lines causes early disruption to cell cycle progression.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Cholera toxin regulates a signaling pathway critical for the expansion of neural stem cell cultures from the fetal and adult rodent brains. 20520777

    New mechanisms that regulate neural stem cell (NSC) expansion will contribute to improved assay systems and the emerging regenerative approach that targets endogenous stem cells. Expanding knowledge on the control of stem cell self renewal will also lead to new approaches for targeting the stem cell population of cancers.Here we show that Cholera toxin regulates two recently characterized NSC markers, the Tie2 receptor and the transcription factor Hes3, and promotes the expansion of NSCs in culture. Cholera toxin increases immunoreactivity for the Tie2 receptor and rapidly induces the nuclear localization of Hes3. This is followed by powerful cultured NSC expansion and induction of proliferation both in the presence and absence of mitogen.Our data suggest a new cell biological mechanism that regulates the self renewal and differentiation properties of stem cells, providing a new logic to manipulate NSCs in the context of regenerative disease and cancer.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Adverse effects of reduced oxygen tension on the proliferative capacity of rat kidney and insulin-secreting cell lines involve DNA damage and stress responses. 18692496

    Standard cell culture conditions do not reflect the physiological environment in terms of oxygen tension (20% vs 3%). The effects of lowering oxygen tension on cell proliferation in culture can be beneficial as well as detrimental depending on the cell line studied, but the molecular mechanism underlying such effects is not fully understood. We observed that the proliferative capacity of the rat cell lines NRK and INS-1 was inhibited when cultured under 3% oxygen as compared to 20% oxygen. Suppression of proliferation in NRK cells was accompanied by induction of DNA double strand breaks whereas in INS-1 cells it was accompanied by up-regulation of p53 and p27. Although Sirt1 was up-regulated in both cell lines by 3% oxygen the effects on antioxidant enzymes (MnSOD, CuZnSOD and catalase) were cell line specific. Marked up-regulation of heme oxygenase-1 (HO-1) was detected in both NRK and INS-1 cells when cultured in 3% oxygen. HO-1 expression can be readily induced by exposure to hydrogen peroxide in culture. These results suggest that reduced oxygen tension suppresses the proliferative capacity of these two cell lines through a stress response that is similar to an oxidative stress response but the molecular events that lead to the reduced cell proliferation are cell line specific.
    Document Type:
    Reference
    Product Catalog Number:
    07-131
    Product Catalog Name:
    Anti-Sirt1(Sir2) Antibody

  • Axon myelination and electrical stimulation in a microfluidic, compartmentalized cell culture platform. 22527791

    Axon demyelination contributes to the loss of sensory and motor function following injury or disease in the central nervous system. Numerous reports have demonstrated that myelination can be achieved in neuron/oligodendrocyte co-cultures. However, the ability to selectively treat neuron or oligodendrocyte (OL) cell bodies in co-cultures improves the value of these systems when designing mechanism-based therapeutics. We have developed a microfluidic-based compartmentalized culture system to achieve segregation of neuron and OL cell bodies while simultaneously allowing the formation of myelin sheaths. Our microfluidic platform allows for a high replicate number, minimal leakage, and high flexibility. Using a custom built lid, fit with platinum electrodes for electrical stimulation (10-Hz pulses at a constant 3 V with ~190 kΩ impedance), we employed the microfluidic platform to achieve activity-dependent myelin segment formation. Electrical stimulation of dorsal root ganglia resulted in a fivefold increase in the number of myelinated segments/mm² when compared to unstimulated controls (19.6 ± 3.0 vs. 3.6 ± 2.3 MBP+ segments/mm²). This work describes the modification of a microfluidic, multi-chamber system so that electrical stimulation can be used to achieve increased levels of myelination while maintaining control of the cell culture microenvironment.
    Document Type:
    Reference
    Product Catalog Number:
    AB980