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On-Demand Webinar Available: Cell Freezing Technologies and Disposable Bioreactors Towards a USP Process
Develop a Fully-Closed USP Process: Use Cell Freezing in Bags and SU Bioreactors
  • Recorded on May 22, 2014
  • Duration: 50 minutes
  • A three-dimensional human neural cell culture model of Alzheimer's disease. 25307057

    Alzheimer's disease is the most common form of dementia, characterized by two pathological hallmarks: amyloid-β plaques and neurofibrillary tangles. The amyloid hypothesis of Alzheimer's disease posits that the excessive accumulation of amyloid-β peptide leads to neurofibrillary tangles composed of aggregated hyperphosphorylated tau. However, to date, no single disease model has serially linked these two pathological events using human neuronal cells. Mouse models with familial Alzheimer's disease (FAD) mutations exhibit amyloid-β-induced synaptic and memory deficits but they do not fully recapitulate other key pathological events of Alzheimer's disease, including distinct neurofibrillary tangle pathology. Human neurons derived from Alzheimer's disease patients have shown elevated levels of toxic amyloid-β species and phosphorylated tau but did not demonstrate amyloid-β plaques or neurofibrillary tangles. Here we report that FAD mutations in β-amyloid precursor protein and presenilin 1 are able to induce robust extracellular deposition of amyloid-β, including amyloid-β plaques, in a human neural stem-cell-derived three-dimensional (3D) culture system. More importantly, the 3D-differentiated neuronal cells expressing FAD mutations exhibited high levels of detergent-resistant, silver-positive aggregates of phosphorylated tau in the soma and neurites, as well as filamentous tau, as detected by immunoelectron microscopy. Inhibition of amyloid-β generation with β- or γ-secretase inhibitors not only decreased amyloid-β pathology, but also attenuated tauopathy. We also found that glycogen synthase kinase 3 (GSK3) regulated amyloid-β-mediated tau phosphorylation. We have successfully recapitulated amyloid-β and tau pathology in a single 3D human neural cell culture system. Our unique strategy for recapitulating Alzheimer's disease pathology in a 3D neural cell culture model should also serve to facilitate the development of more precise human neural cell models of other neurodegenerative disorders.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
  • A tissue-like construct of human bone marrow MSCs composite scaffold support in vivo ectopic bone formation. 19842114

    Biocompatible and osteoconductive cell-scaffold constructs comprise the first and most important step towards successful in vivo bone repair. This study reports on a new cell-scaffold construct composed of gelatin-based hydrogel and ceramic (CaCO(3)/beta-TCP) particles loaded with human MSCs producing a tissue-like construct applied as a transplant for in vivo bone formation. Bone marrow-derived human MSCs were cultured in osteogenic induction medium. 5 x 10(5) (P(2)) cells were loaded on a mixture of hydrogel microspheres and ceramic particles, cultured in a rotating dynamic culture for up to 3 weeks. Both hydrogel microspheres and ceramic particles coalesced together to form a tissue-like construct, shown by histology to contain elongated spindle-like cells forming the new tissue between the individual particles. Cell proliferation and cell viability were confirmed by Alamar blue assay and by staining with CFDA, respectively. FACS analysis conducted before loading the cells, and after formation of the construct, revealed that the profile of cell surface markers remained unchanged throughout the dynamic culture. The osteogenic potential of the cells composing the tissue-like construct was further validated by subcutaneous transplants in athymic nude mice. After 8 weeks a substantial amount of new bone formation was observed in the cell-construct transplants, whereas no bone formation was observed in transplants containing no cells. This new cell construct provides a system for in vivo bone transplants. It can be tailored for a specific size and shape as needed for various transplant sites and for all aspects of regenerative medicine and biomaterial science.
    Document Type:
    Reference
    Product Catalog Number:
    CBL415F
  • A total water purification system A total water purification system

    Many of the analytical and molecular biology applications that require the use of water include high-performance liquid chromatography (HPLC), total organic carbon (TOC) analysis, sample and media preparation, rinse steps in assays, and gel electrophoresis. Different types of laboratories run experiments that require varying levels of water purity. What is needed in one lab might not be needed in another. Therefore, professional organizations have established water quality standards or guidelines to facilitate laboratory water purification within various industry sectors
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
  • A widely expressed transcription factor with multiple DNA sequence specificity, CTCF, is localized at chromosome segment 16q22.1 within one of the smallest regions of ove ... 9591631

    The cellular protooncogene MYC encodes a nuclear transcription factor that is involved in regulating important cellular functions, including cell cycle progression, differentiation, and apoptosis. Dysregulated MYC expression appears critical to the development of various types of malignancies, and thus factors involved in regulating MYC expression may also play a key role in the pathogenesis of certain cancers. We have cloned one such MYC regulatory factor, termed CTCF, which is a highly evolutionarily conserved-11-zinc finger transcriptional factor possessing multiple DNA sequence specificity. CTCF binds to a number of important regulatory regions within the 5' noncoding sequence of the human MYC oncogene, and it can regulate its transcription in several experimental systems. CTCF mRNA is expressed in cells of multiple different lineages. Enforced ectopic expression of CTCF inhibits cell growth in culture. Southern blot analyses and fluorescence in situ hybridization (FISH) with normal human metaphase chromosomes showed that the human CTCF is a single-copy gene situated at chromosome locus 16q22. Cytogenetic studies have pointed out that chromosome abnormalities (deletions) at this locus frequently occur in many different human malignancies, suggesting the presence of one or more tumor suppressor genes in the region. To narrow down their localization, several loss of heterozygosity (LOH) studies of chromosome arm 16q in sporadic breast and prostate cancers have been carried out to define the most recurrent and smallest region(s) of overlap (SRO) for commonly deleted chromosome arm 16q material. For CTCF to be considered as a candidate tumor suppressor gene associated with tumorigenesis, it should localize within one of the SROs at 16q. Fine-mapping of CTCF has enabled us to assign the CTCF gene to about a 2 centiMorgan (cM) interval of 16q22.1 between the somatic cell hybrid breakpoints CY130(D) and CY4, which is between markers D16S186 (16AC16-101) and D16S496 (AFM214zg5). This relatively small region, containing the CTCF gene, overlaps the most frequently observed SROs for common chromosomal deletions found in sporadic breast and prostate tumors. In one of four analyzed paired DNA samples from primary breast cancer patients, we have detected a tumor-specific rearrangement of CTCF exons encoding the 11-zinc-finger domain. Therefore, taken together with other CTCF properties, localization of CTCF to a narrow cancer-associated chromosome region suggests that CTCF is a novel candidate tumor suppressor gene at 16q22.1.
    Document Type:
    Reference
    Product Catalog Number:
    07-729
    Product Catalog Name:
    Anti-CTCF Antibody
  • Aberrant activation of the interleukin-2 autocrine loop through the nuclear factor of activated T cells by nonleukemogenic human T-cell leukemia virus type 2 but not by l ... 16140768

    Human T-cell leukemia virus type 1 (HTLV-1) but not HTLV-2 is associated with adult T-cell leukemia. We found that HTLV-2 Tax2 protein stimulated reporter gene expression regulated by the interleukin (IL)-2 promoter through the nuclear factor of activated T cells (NFAT) in a human T-cell line (Jurkat). However, the activity of HTLV-1 Tax1 was minimal in this system. T-cell lines immortalized by HTLV-2 but not HTLV-1 constitutively exhibited activated NFAT in the nucleus and constitutively expressed IL-2 mRNA. Cyclosporine A, an inhibitor of NFAT activation, abrogated the induction of IL-2 mRNA in HTLV-2-immortalized T-cell lines and concomitantly inhibited cell growth. This growth inhibition was rescued by the addition of IL-2 to the culture. Furthermore, anti-IL-2 receptor antibodies significantly reduced the proliferation of HTLV-2-infected T-cell lines but not that of HTLV-1-infected cells. Our results suggest that Tax2 activates an IL-2 autocrine loop mediated through NFAT that supports the growth of HTLV-2-infected cells under low-IL-2 conditions. This mechanism would be especially important in vivo, where this autocrine mechanism establishes a nonleukemogenic life-long HTLV-2 infection. The results also suggest that differences in long-term cytokine production between HTLV-1 and HTLV-2 infection are another factor for the differences in pathogenesis.
    Document Type:
    Reference
    Product Catalog Number:
    PP102
    Product Catalog Name:
    IgG2a Antibody, Mouse
  • Aberrant epigenetic silencing is triggered by a transient reduction in gene expression. 19279688

    Aberrant epigenetic silencing plays a major role in cancer formation by inactivating tumor suppressor genes. While the endpoints of aberrant silencing are known, i.e., promoter region DNA methylation and altered histone modifications, the triggers of silencing are not known. We used the tet-off system to test the hypothesis that a transient reduction in gene expression will sensitize a promoter to undergo epigenetic silencing.The tet responsive promoter (P(TRE)) was used to drive expression of the selectable human HPRT cDNA in independent transfectants of an Hprt deficient mouse cell line. In this system, high basal HPRT expression is greatly reduced when doxycycline (Dox) is added to the culture medium. Exposure of the P(TRE)-HPRT transfectants to Dox induced HPRT deficient clones in a time dependent manner. A molecular analysis demonstrated promoter region DNA methylation, loss of histone modifications associated with expression (i.e., H3 lysine 9 and 14 acetylation and lysine 4 methylation), and acquisition of the repressive histone modification H3 lysine 9 methylation. These changes, which are consistent with aberrant epigenetic silencing, were not present in the Dox-treated cultures, with the exception of reduced H3 lysine 14 acetylation. Silenced alleles readily reactivated spontaneously or after treatment of cells with inhibitors of histone deacetylation and/or DNA methylation, but re-silencing of reactivated alleles did not require a new round of Dox exposure. Inhibition of histone deacetylation inhibited both the induction of silencing and re-silencing, whereas inhibition of DNA methylation had no such effect.This study demonstrates that a transient reduction in gene expression triggers a pathway for aberrant silencing in mammalian cells and identifies histone deacetylation as a critical early step in this process. DNA methylation, in contrast, is a secondary step in the silencing pathway under study. A model to explain these observations is offered.
    Document Type:
    Reference
    Product Catalog Number:
    17-371
    Product Catalog Name:
    EZ-ChIP™
  • Aberrant expression of corticotropin-releasing hormone in pre-eclampsia induces expression of FasL in maternal macrophages and extravillous trophoblast apoptosis. 22763913

    Corticotropin-releasing hormone (CRH) and its receptors are expressed in human placenta. Recently, the impaired function of this system has been associated with a number of complications of pregnancy, including pre-eclampsia. The aim of the study was to test the hypothesis that CRH participates in the pathophysiology of pre-eclampsia through the induction of macrophage-mediated apoptosis of extravillous trophoblasts (EVTs). We found that the expression of CRH was increased in the EVT of the placental bed biopsy specimens from pre-eclamptic pregnancies (1.8-fold increase; P < 0.05). In addition, significantly larger numbers of apoptotic EVT were detected in pre-eclamptic placentas compared with normal ones (P < 0.05), and only in pre-eclamptic placentas, decidual macrophages were found to be Fas ligand (FasL)-positive. In vitro studies on the effect of CRH on human macrophages suggested that CRH induced the expression of the FasL protein in human macrophages and potentiated their ability to induce the apoptosis of a Fas-expressing EVT-based hybridoma cell line in co-cultures. These findings demonstrate a possible mechanism by which the aberrant expression of CRH in pre-eclampsia may activate the FasL-positive decidual macrophages, impair the physiological turnover of EVT and eventually disturb placentation.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1501
    Product Catalog Name:
    Anti-Actin Antibody, clone C4
  • AC927, a σ receptor ligand, blocks methamphetamine-induced release of dopamine and generation of reactive oxygen species in NG108-15 cells. 22101517

    Methamphetamine is a highly addictive psychostimulant drug of abuse that causes neurotoxicity with high or repeated dosing. Earlier studies demonstrated the ability of the selective σ receptor ligand N-phenethylpiperidine oxalate (AC927) to attenuate the neurotoxic effects of methamphetamine in vivo. However, the precise mechanisms through which AC927 conveys its protective effects remain to be determined. With the use of differentiated NG108-15 cells as a model system, the effects of methamphetamine on neurotoxic endpoints and mediators such as apoptosis, necrosis, generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), and dopamine release were examined in the absence and presence of AC927. Methamphetamine at physiologically relevant micromolar concentrations caused apoptosis in NG108-15 cells. At higher concentrations of methamphetamine, necrotic cell death was observed. At earlier time points, methamphetamine caused ROS/RNS generation, which was detected with the fluorigenic substrate 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescin diacetate, acetyl ester, in a concentration- and time-dependent manner. N-Acetylcysteine, catalase, and l-N(G)-monomethyl arginine citrate inhibited the ROS/RNS fluorescence signal induced by methamphetamine, which suggests the formation of hydrogen peroxide and RNS. Exposure to methamphetamine also stimulated the release of dopamine from NG108-15 cells into the culture medium. AC927 attenuated methamphetamine-induced apoptosis, necrosis, ROS/RNS generation, and dopamine release in NG108-15 cells. Together, the data suggest that modulation of σ receptors can mitigate methamphetamine-induced cytotoxicity, ROS/RNS generation, and dopamine release in cultured cells.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
  • Accelerated repair of demyelinated CNS lesions in the absence of non-muscle myosin IIB. 24470341

    The oligodendrocyte (OL), the myelinating cell of the central nervous system, undergoes dramatic changes in the organization of its cytoskeleton as it differentiates from a precursor (oligodendrocyte precursor cells) to a myelin-forming cell. These changes include an increase in its branching cell processes, a phenomenon necessary for OL to myelinate multiple axon segments. We have previously shown that levels and activity of non-muscle myosin II (NMII), a regulator of cytoskeletal contractility, decrease as a function of differentiation and that inhibition of NMII increases branching and myelination of OL in coculture with neurons. We have also found that mixed glial cell cultures derived from NMIIB knockout mice display an increase in mature myelin basic protein-expressing OL compared with wild-type cultures. We have now extended our studies to investigate the role of NMIIB ablation on myelin repair following focal demyelination by lysolecithin. To this end, we generated an oligodendrocyte-specific inducible knockout model using a Plp-driven promoter in combination with a temporally activated CRE-ER fusion protein. Our data indicate that conditional ablation of NMII in adult mouse brain, expedites lesion resolution and remyelination by Plp+ oligodendrocyte-lineage cells when compared with that observed in control brains. Taken together, these data validate the function of NMII as that of a negative regulator of OL myelination in vivo and provide a novel target for promoting myelin repair in conditions such as multiple sclerosis.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
  • Accumulation of acetylcholine receptors is a necessary condition for normal accumulation of acetylcholinesterase during in vitro neuromuscular synaptogenesis. 9751136

    To study a step of the very complex processes of the formation of the neuromuscular junction (NMJ), we have analysed the clustering of acetylcholine receptors (AChR) and acetylcholinesterase (AChE) in myotubes cultured in various conditions. On the surface of rat myotubes cultured in the presence of spinal cord cells from embryonic rat, numerous AChE clusters appeared. Such clusters are always co-localized with AChR clusters, but the reverse is not true: the number of AChR clusters largely exceeds that of AChE clusters. Very few AChE clusters formed when such co-cultures were treated with monoclonal antibodies (mAbs) against the main immunogenic region (MIR) of the AChR, which provoke internalization and degradation of the AChRs of the muscular membrane. The total levels of AChE and proportions of molecular forms were unaffected. We also used non-innervated myotubes in which addition of agrin, a protein normally synthesized by motoneurons, transported to nerve terminals and inserted into the synaptic basal lamina, induces the formation of small clusters of AChE. When added to rat myotubes devoid of membrane AChR, agrin-induced AChE clusters did not form. Finally, we analysed the capacity of the variant of the C2 mouse muscle cell line deficient in AChR (1R-) to form clusters of AChE in co-cultures with spinal cord cells from rat: no formation of AChE clusters could be observed. In all these different systems of cultures, the conditions which prevented clustering of AChR (anti-AChR antibodies, deficiency of the variant C2 cell line) also suppressed AChE clustering. We concluded that clustering of AChR is a prerequisite for clustering of AChE, so that NMJ formation implies the sequential accumulation of these two components.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1951F
    Product Catalog Name:
    Anti-Integrin β1 Antibody, clone P4G11, FITC conjugated