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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

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Recorded on May 22, 2014
Duration: 50 minutes

Developmental changes of parameters for astrogliosis during cultivation of purified cerebral astrocytes from newborn rats.
Astrogliosis is a common phenomenon seen in most neuropathological changes of the central nervous system. Several in vitro models have been used to study the mechanisms and conditions for the induction of astrogliosis, however many do not take into account that the metabolic and structural characteristics of astrocytes change with time in culture. Thus, it appears difficult to attribute changes of, e.g., GFAP to the normal change in vitro as opposed to additional changes due to an astrogliotic reaction. The present study was therefore undertaken to characterize these developmental changes in purified astroglial secondary cultures during cultivation to provide a basis for further investigations of astrogliosis in vitro. During 6 weeks of cultivation (3-43 days) GFAP (ELISA) increased much more (22-fold) than the cell number (2.5-fold) and the total protein (3.5-fold). The GFAP/protein ratio increased during the first 4 weeks of cultivation and reached a plateau thereafter, which was accompanied by a significant increase of GFAP mRNA (Northern blot). At the ultrastructural level (transmission electron microscopy) gliofilaments in the perinuclear region as well as in the cell processes of 4-day-old astrocytes showed a dispersed pattern, whereas an accumulation of gliofilaments was found in 39-day-old cells, which formed large aggregated bundles localized mostly in the cell processes. Our results show that in vitro astrocytes undergo developmental changes in their accumulation of GFAP and intermediate filaments which reach a stable steady state after 4 weeks in culture. These 'normal' developmental changes will have to be taken into account, when experiments with variations of the level of GFAP are performed. Stable culture conditions for experimentation appear to be present after 4 weeks in culture.
Document Type:

Reference

Product Catalog Number:

MAB3402
Product Catalog Name:

Anti-Glial Fibrillary Acidic Protein Antibody, clone GA5
Developmental expression of BMP4/ALK3/SMAD5 signaling pathway in the mouse testis: a potential role of BMP4 in spermatogonia differentiation.
It is well established that the c-kit gene plays an essential role in the proliferation of differentiating spermatogonia in prepuberal mice. However, the mechanisms that regulate the onset of spermatogenesis, i.e. differentiation of spermatogonial stem cells and c-kit expression, are poorly understood. Here we identify a novel signal transduction system in mouse prepuberal testis regulating this developmental event, involving bone morphogenetic protein 4 (BMP4) and its transduction machinery. BMP4 is produced by Sertoli cells very early in the postnatal life and is successively down regulated in peri-puberal Sertoli cells. Its receptor Alk3 and the R-Smad Smad5 are specifically expressed both in proliferating primordial germ cells and in postnatal spermatogonia. BMP4 stimulation of cultured spermatogonia induces Smad4/5 nuclear translocation and the formation of a DNA-binding complex with the transcriptional coactivator p300/CBP. In vitro exposure of undifferentiated spermatogonia to BMP4 exerts both mitogenic and differentiative effects, inducing [3H]thymidine incorporation and Kit expression. As a result of the latter event, Kit-negative spermatogonia acquire sensitivity to Stem Cell Factor.
Document Type:

Reference

Product Catalog Number:

06-755
Product Catalog Name:

Anti-Histone H3 Antibody
Developmental switch in axon guidance modes of hippocampal mossy fibers in vitro.
Hippocampal mossy fibers (MFs), axons of dentate granule cells, run through a narrow strip, called the stratum lucidum, and make synaptic contacts with CA3 pyramidal cells. This stereotyped pathfinding is assumed to require a tightly controlled guidance system, but the responsible mechanisms have not been proven directly. To clarify the cellular basis for the MF pathfinding, microslices of the dentate gyrus (DG) and Ammon's horn (AH) were topographically arranged in an organotypic explant coculture system. When collagen gels were interposed between DG and AH slices prepared from postnatal day 6 (P6) rats, the MFs passed across this intervening gap and reached CA3 stratum lucidum. Even when the recipient AH was chemically pre-fixed with paraformaldehyde, the axons were still capable of accessing their normal target area only if the DG and AH slices were directly juxtaposed without a collagen bridge. The data imply that diffusible and contact cues are both involved in MF guidance. To determine how these different cues contribute to MF pathfinding during development, a P6 DG slice was apposed simultaneously to two AH slices prepared from P0 and P13 rats. MFs projected normally to both the host slices, whereas they rarely invaded P0 AH when the two hosts were fixed. Early in development, therefore, the MFs are guided mainly by a chemoattractant gradient, and thereafter, they can find their trajectories by a contact factor, probably via fasciculation with pre-established MFs. The present study proposes a dynamic paradigm in CNS axon pathfinding, that is, developmental changes in axon guidance cues.
Document Type:

Reference

Product Catalog Number:

Multiple
Product Catalog Name:

Multiple
Differential control of Wnt target genes involves epigenetic mechanisms and selective promoter occupancy by T-cell factors.
Canonical Wnt signaling and its nuclear effectors, beta-catenin and the family of T-cell factor (TCF) DNA-binding proteins, belong to the small number of regulatory systems which are repeatedly used for context-dependent control of distinct genetic programs. The apparent ability to elicit a large variety of transcriptional responses necessitates that beta-catenin and TCFs distinguish precisely between genes to be activated and genes to remain silent in a specific context. How this is achieved is unclear. Here, we examined patterns of Wnt target gene activation and promoter occupancy by TCFs in different mouse cell culture models. Remarkably, within a given cell type only Wnt-responsive promoters are bound by specific subsets of TCFs, whereas nonresponsive Wnt target promoters remain unoccupied. Wnt-responsive, TCF-bound states correlate with DNA hypomethylation, histone H3 hyperacetylation, and H3K4 trimethylation. Inactive, nonresponsive promoter chromatin shows DNA hypermethylation, is devoid of active histone marks, and additionally can show repressive H3K27 trimethylation. Furthermore, chromatin structural states appear to be independent of Wnt pathway activity. Apparently, cell-type-specific regulation of Wnt target genes comprises multilayered control systems. These involve epigenetic modifications of promoter chromatin and differential promoter occupancy by functionally distinct TCF proteins, which together determine susceptibility to Wnt signaling.
Document Type:

Reference

Product Catalog Number:

Multiple
Product Catalog Name:

Multiple
Differential miRNA expression profiles in proliferating or differentiated keratinocytes in response to gamma irradiation.
MicroRNAs (miRNAs), a group of short non-coding RNAs that negatively regulate gene expression, have recently emerged as potential modulators of cellular response to ionizing radiations both in vitro and in vivo in various cell types and tissues. However, in epidermal cells, the involvement of the miRNA machinery in the cellular response to ionizing radiations remains to be clarified. Indeed, understanding the mechanisms of cutaneous radiosensitivity is an important issue since skin is the most exposed organ to ionizing radiations and among the most sensitive.We settled up an expression study of miRNAs in primary human skin keratinocytes using a microfluidic system of qPCR assay, which permits to assess the expression of almost 700 annotated miRNAs. The keratinocytes were cultured to a proliferative or a differentiated state mimicking basal or suprabasal layers of human epidermis. These cells were irradiated at 10 mGy or 6 Gy and RNA was extracted 3 hours after irradiation. We found that proliferative cells irradiated at 6 Gy display a global fall of miRNA expression whereas differentiated cells exposed to the same dose display a global increase of miRNAs expression. We identified twenty miRNAs weakly but significantly modulated after 6 Gy irradiation, whereas only 2 miRNAs were modulated after low-dose irradiation in proliferating cells. To go further into the biological meaning of this miRNA response, we over-expressed some of the responding miRNA in proliferating cells: we observed a significant decrease of cell viability 72 hours after irradiation. Functional annotation of their predicted targets revealed that G-protein related pathways might be regulated by these responding miRNAs.Our results reveal that human primary keratinocytes exposed to ionizing irradiation expressed a miRNA pattern strongly related to the differentiation status of irradiated cells. We also demonstrate that some miRNAs play a role in the radiation response to ensure the short-term survival of irradiated keratinocytes.
Document Type:

Reference

Product Catalog Number:

MAB1501
Product Catalog Name:

Anti-Actin Antibody, clone C4
Differential modulation of basic fibroblast and epidermal growth factor receptor activation by ganglioside GM3 in cultured retinal Müller glia.
Polypeptide growth factors and membrane-bound gangliosides are involved in cell signaling, including that observed in cells of neural origin. To analyze possible interactions between these two systems, we investigated the modulation of short- and long-term responses to basic fibroblast and epidermal growth factor (bFGF and EGF, respectively) in cultured retinal Müller glial cells following experimental modification of their ganglioside composition. These glial cells readily incorporated exogenously administered GM3 ganglioside, which was not substantially metabolized within 24 h. Such treatments significantly inhibited bFGF-induced DNA replication and cell migration, while having much less effect on analogous EGF-mediated behaviors. To explore GM3/growth factor interactions further, different aspects of glial metabolism in response to bFGF or EGF stimulation were examined: membrane fluidity, growth factor binding, global and individual changes in growth factor-induced phosphotyrosine levels, and growth factor-induced activation of mitogen-activated protein kinase. GM3 reduced the intensity of immunocytochemical labeling of phosphotyrosine-containing proteins within bFGF-stimulated cells and down-regulated FGF receptor activation and tyrosine phosphorylation of its cellular substrates, whereas similar parameters in EGF-stimulated cells were much less affected. Hence the data reveal a complex relationship in normal neural cells between polypeptide growth factors and membrane-bound gangliosides, which may participate in retinal cellular physiology in vivo.
Document Type:

Reference

Product Catalog Number:

Multiple
Product Catalog Name:

Multiple
Differential responses of healthy and chronic obstructive pulmonary diseased human bronchial epithelial cells repeatedly exposed to air pollution-derived PM4.
While the knowledge of the underlying mechanisms by which air pollution-derived particulate matter (PM) exerts its harmful health effects is still incomplete, detailed in vitro studies are highly needed. With the aim of getting closer to the human in vivo conditions and better integrating a number of factors related to pre-existing chronic pulmonary inflammatory, we sought to develop primary cultures of normal human bronchial epithelial (NHBE) cells and chronic obstructive pulmonary disease (COPD)-diseased human bronchial epithelial (DHBE) cells, grown at the air-liquid interface. Pan-cytokeratin and MUC5AC immunostaining confirmed the specific cell-types of both these healthy and diseased cell models and showed they are closed to human bronchial epithelia. Thereafter, healthy and diseased cells were repeatedly exposed to air pollution-derived PM4 at the non-cytotoxic concentration of 5 μg/cm2. The differences between the oxidative and inflammatory states in non-exposed NHBE and COPD-DHBE cells indicated that diseased cells conserved their specific physiopathological characteristics. Increases in both oxidative damage and cytokine secretion were reported in repeatedly exposed NHBE cells and particularly in COPD-DHBE cells. Diseased cells repeatedly exposed had lower capacities to metabolize the organic chemicals-coated onto the air-pollution-derived PM4, such as benzo[a]pyrene (B[a]P), but showed higher sensibility to the formation of OH-B[a]P DNA adducts, because their diseased state possibly affected their defenses. Differential profiles of epigenetic hallmarks (i.e., global DNA hypomethylation, P16 promoter hypermethylation, telomere length shortening, telomerase activation, and histone H3 modifications) occurred in repeatedly exposed NHBE and particularly in COPD-DHBE cells. Taken together, these results closely supported the highest responsiveness of COPD-DHBE cells to a repeated exposure to air pollution-derived PM4. The use of these innovative in vitro exposure systems such as NHBE and COPD-DHBE cells could therefore be consider as a very useful and powerful promising tool in the field of the respiratory toxicology, taking into account sensitive individuals.
Document Type:

Reference

Product Catalog Number:

17-10451
Product Catalog Name:

CpGenome Direct Prep Bisulfite Modification Kit (50 Reactions)
Differential utilization of VLA-4 (alpha 4 beta 1) and -5 (alpha 5 beta 1) integrins during the development of mouse bone marrow-derived mast cells.
Cytokines have been shown to have major roles in the development of mast cells from bone marrow progenitors. Immature mast cells derived from bone marrow thus leave the blood system to complete their course of maturation within tissues. However, it is now clear that VLA (beta 1) integrins with function in mediating cell-cell and cell-extracellular matrix protein interactions have effects on the growth and differentiation of diverse cell types. At present, the involvement of VLA integrins during mast cell development is still unclear. In this study, we report the preparation of a new monoclonal antibody (mAb) against mouse VLA-5 (alpha 5 beta 1) integrin. Together with mAb R1-2, we characterized the expression of VLA-4 (alpha 4 beta 1) and VLA-5 integrins, the two major fibronectin receptors, on two long-term cultured mast cell lines, CFTL-15 and MC/9. CFTL-15 cells were found to express both VLA-4 and -5 integrins whereas MC/9 cells expressed only VLA-5 but not VLA-4. We speculated that VLA integrin expression may be related to mast cell development. Thus bone marrow-derived mast cells (BMMC) were characterized after varying periods of development induced by IL-3. During the first 3 weeks the expression of VLA-4 and VLA-5 increased progressively and both were involved in mediating adhesion of BMMC to fibronectin. At time periods of greater than 3 weeks, the expression of VLA-4 declined gradually to little, if any, by week 13. In comparison, VLA-5 remained stably expressed and functioned as the major receptor for fibronectin. Results from this study therefore suggest that BMMC differentially utilize VLA-4 and VLA-5 integrins during IL-3-induced development. Differential expression of VLA integrins may have effects on the recirculation properties, tissue distribution and eventual maturation of progenitors to fully matured mast cells.
Document Type:

Reference

Product Catalog Number:

MAB1984-I
Product Catalog Name:

Anti-Integrin α5β1 Antibody, clone BMA5
Differentiated human neural stem cells: a new ex vivo model to study HHV-6 infection of the central nervous system.
BACKGROUND: HHV-6 is the etiologic agent of exanthem subitum, a pediatric illness that may be associated with clinical and laboratory signs of central nervous system involvement. The absence of suitable experimental models has so far hampered the elucidation of the mechanisms of HHV-6-mediated neural cell damage. Recently, the growing knowledge in neurobiology has permitted the establishment of long-term cultures of human neural stem cells (hNSC) that, by virtue of their self-renewal capacity and multipotentiality, provide a valuable tool for the study of neurodegenerative disorders. OBJECTIVES AND STUDY DESIGN: We studied the effects of HHV-6 infection in differentiated cultures of hNSC derived from the telencephalic and diencephalic regions of a 13.5 week post conception (pcw) fetal brain. The prototypic HHV-6 strain GS (subgroup A) was used. RESULTS: hNSC were differentiated ex vivo to obtain mixed cultures encompassing astrocytes, neurons and oligodendrocytes. These differentiated hNSC cultures were found to be susceptible to productive HHV-6A infection, resulting in the formation of syncytia associated with phenotypic alterations. CONCLUSION: These results demonstrate that hNSC may provide a physiologically relevant model to investigate the pathogenic role of HHV-6 in central nervous system disorders.
Document Type:

Reference

Product Catalog Number:

MAB345
Product Catalog Name:

Anti-O4 Antibody, clone 81
Differentiation of glial cells and motor neurons during the formation of neuromuscular junctions in cocultures of rat spinal cord explant and human muscle.
Motor axons extending from embryonic rat spinal cord explants form fully mature neuromuscular junctions with cocultured human muscle. This degree of maturation is not observed in muscle innervated by dissociated motor neurons. Glial cells present in the spinal cord explants seem to be, besides remaining interneurons, the major difference between the two culture systems. In light of this observation and the well documented role of glia in neuronal development, it can be hypothesized that differentiated and long-lived neuromuscular junctions form in vitro only if their formation is accompanied by codifferentiation of neuronal and glial cells and if this codifferentiation follows the spatial and temporal pattern observed in vivo. Investigation of this hypothesis necessitates the characterization of neuronal and glial cell development in spinal cord explant-muscle cocultures. No such study has been reported, although these cocultures have been used in numerous studies of neuromuscular junction formation. The aim of this work was therefore to investigate the temporal relationship between neuromuscular junction formation and the differentiation of neuronal and glial cells during the first 3 weeks of coculture, when formation and development of the neuromuscular junction occurs in vitro. The expression of stage-specific markers of neuronal and glial differentiation in these cocultures was characterized by immunocytochemical and biochemical analyses. Differentiation of astrocytes, Schwann cells, and oligodendrocytes proceeded in concert with the differentiation of motor neurons and neuromuscular junction formation. The temporal coincidence between maturation of the neuromuscular junction and lineage progression of neurons and glial cells was similar to that observed in vivo. These findings support the hypothesis that glial cells are a major contributor to maturity of the neuromuscular junction formed in vitro in spinal cord explant-muscle cocultures.
Document Type:

Reference

Product Catalog Number:

AB1783
Product Catalog Name:

Anti-Glutamate Transporter Antibody, Glial