搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

The screening for antigen-specific hybridoma cells with adequate production rates is still a time-,labour- and money-consuming procedure. A reduction in cell culture testing by specifically selecting those fused cells that produce antibody could therefore make hybridoma technology more attractive,even for small research groups or for newly discovered proteins at an early stage of research. Additional problems,such as the requirement to produce sufficient amounts of the unknown protein at a purity that allows specific immunisation of mice and testing of the resulting hybridoma clones,also need to be overcome. Here we present a new strategy to isolate rapidly and efficiently monoclonal antibodies against new proteins,for which only sequence information at the DNA level is known. The strategy consists of fusion of the protein to a hexa-His-tag to allow easy purification,production in yeast and insect cells to reduce background immunisation with host cell proteins and the selection of IgG-producing hybridoma cells by flow-cytometric cell sorting using the affinity matrix secretion assay technique. ?? 2005 Elsevier B.V. All rights reserved. View Publication

CD24 expression on pro-B cells plays a role in B cell selection and development in the bone marrow. We previously detected higher CD24 expression and frequency within IgD+ na{\{i}}ve and memory B cells in patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) compared with age-matched healthy controls (HC). Here we investigated the relationship between CD24 expression and B cell maturation. In vitro stimulation of isolated B cells in response to conventional agonists were used to follow the dynamics of CD24 positivity during proliferation and differentiation (or maturation). The relationship between CD24 expression to cycles of proliferation and metabolism in purified B cells from HC was also investigated using phospho-flow (phosphorylation of AMPK-pAMPK) 1proton nuclear magnetic resonance and Mitotracker Far-red (Mitochondrial mass-MM). In vitro in the absence of stimulation there was an increased percentage of CD24+ viable B cells in ME/CFS patients compared to HC (p {\textless} 0.05) following 5 days culture. Following stimulation with B cell agonists percentage of CD24+B cells in both na{\"{i}}ve and memory B cell populations decreased. P {\textless} 0.01). There was a negative relationship between percentage of CD24+B cells with MM (R2 = 0.76; p {\textless} 0.01) which was subsequently lost over sequential cycles of proliferation. There was a significant correlation between CD24 expression on B cells and the usage of glucose and secretion of lactate in vitro. Short term ligation of the B cell receptor with anti-IgM antibody significantly reduced the viability of CD24+ memory B cells compared to those cross-linked by anti-IgD or anti-IgG antibody. A clear difference was found between na{\""{i}}ve and memory B cells with respect to CD24 expression and pAMPK most notably a strong positive association in IgD+IgM+ memory B cells. In vitro findings confirmed dysregulation of CD24-expressing B cells from ME/CFS patients previously suggested by immunophenotype studies of B cells from peripheral blood. CD24-negative B cells underwent productive proliferation whereas CD24+ B cells were either unresponsive or susceptible to cell death upon BCR-engagement alone. We suggest that CD24 expression may reflect variations in energy metabolism on different B cell subsets.""" View Publication

Haploid human pluripotent stem cells (PSCs) integrate haploidy and pluripotency,providing a novel system for functional genomics and developmental research in humans. We have recently derived haploid human embryonic stem cells (ESCs) by parthenogenesis and demonstrated their wide differentiation potential and applicability for genetic screening. Because haploid cells can spontaneously become diploid,their enrichment at an early passage is key for successful derivation. In this protocol,we describe two methodologies,namely metaphase spread analysis and cell sorting,for the identification of haploid human cells within parthenogenetic ESC lines. The cell sorting approach also enables the isolation of haploid cells at low percentages,as well as the maintenance of highly enriched haploid ESC lines throughout passaging. The isolation of essentially pure populations of haploid human ESCs by this protocol requires basic PSC culture expertise and can be achieved within 4-6 weeks. View Publication

Elucidation of the cellular and molecular mechanisms that maintain mammary epithelial tissue integrity is of broad interest and paramount to the design of more effective treatments for breast cancer. Evidence from both in vitro and in vivo experiments suggests that mammary cell differentiation is a hierarchical process originating in an uncommitted stem cell with self-renewal potential. However,analysis of the properties and regulation of mammary stem cells has been limited by a lack of methods for their prospective isolation. Here we report the use of multi-parameter cell sorting and limiting dilution transplant analysis to demonstrate the purification of a rare subset of adult mouse mammary cells that are able individually to regenerate an entire mammary gland within 6 weeks in vivo while simultaneously executing up to ten symmetrical self-renewal divisions. These mammary stem cells are phenotypically distinct from and give rise to mammary epithelial progenitor cells that produce adherent colonies in vitro. The mammary stem cells are also a rapidly cycling population in the normal adult and have molecular features indicative of a basal position in the mammary epithelium. View Publication

Human brain development exhibits several unique aspects,such as increased complexity and expansion of neuronal output,that have proven difficult to study in model organisms. As a result,in vitro approaches to model human brain development and disease are an intense area of research. Here we describe a recently established protocol for generating 3D brain tissue,so-called cerebral organoids,which closely mimics the endogenous developmental program. This method can easily be implemented in a standard tissue culture room and can give rise to developing cerebral cortex,ventral telencephalon,choroid plexus and retinal identities,among others,within 1-2 months. This straightforward protocol can be applied to developmental studies,as well as to the study of a variety of human brain diseases. Furthermore,as organoids can be maintained for more than 1 year in long-term culture,they also have the potential to model later events such as neuronal maturation and survival. View Publication

Human pluripotent stem cells (hPSCs) are sensitive to DNA damage and undergo rapid apoptosis compared to their differentiated progeny cells. Here,we explore the underlying mechanisms for the increased apoptotic sensitivity of hPSCs that helps to determine pluripotent stem cell fate. Apoptosis was induced by exposure to actinomycin D,etoposide,or tunicamycin,with each agent triggering a distinct apoptotic pathway. We show that hPSCs are more sensitive to all three types of apoptosis induction than are lineage-non-specific,retinoic-acid-differentiated hPSCs. Also,Bax activation and pro-apoptotic mitochondrial intermembrane space protein release,which are required to initiate the mitochondria-mediated apoptosis pathway,are more rapid in hPSCs than in retinoic-acid-differentiated hPSCs. Surprisingly,Bak and not Bax is essential for actinomycin-D-induced apoptosis in human embryonic stem cells. Finally,P53 is degraded rapidly in an ubiquitin-proteasome-dependent pathway in hPSCs at steady state but quickly accumulates and induces apoptosis when Mdm2 function is impaired. Rapid degradation of P53 ensures the survival of healthy hPSCs but avails these cells for immediate apoptosis upon cellular damage by P53 stabilization. Altogether,we provide an underlying,interconnected molecular mechanism that primes hPSCs for quick clearance by apoptosis to eliminate hPSCs with unrepaired genome alterations and preserves organismal genomic integrity during the early critical stages of human embryonic development. View Publication

Human pluripotent stem cells (hPSCs) can self-renew or differentiate to diverse cell types,thus providing a platform for basic and clinical applications. However,pluripotent stem cell populations are heterogeneous and functional properties at the single cell level are poorly documented leading to inefficiencies in differentiation and concerns regarding reproducibility and safety. Here,we use non-invasive time-lapse imaging to continuously examine hPSC maintenance and differentiation and to predict cell viability and fate. We document dynamic behaviors and social interactions that prospectively distinguish hPSC survival,self-renewal,and differentiation. Results highlight the molecular role of E-cadherin not only for cell-cell contact but also for clonal propagation of hPSCs. Results indicate that use of continuous time-lapse imaging can distinguish cellular heterogeneity with respect to pluripotency as well as a subset of karyotypic abnormalities whose dynamic properties were monitored. View Publication

Pig induced pluripotent stem cells (piPSCs) offer a great opportunity and a number of advantages in the generation of transgenic animals. These immortalized cells can undergo multiple rounds of genetic modifications (e.g.,gene knock-in,knockout) and selection leading to animals that have optimized traits of biomedical or agricultural interests. In this chapter we describe the production and characterization of piPSCs,microinjection of piPSCs into embryos,embryo transfer and production of chimeric animals based on successful protocols. View Publication

Diploidy is a fundamental genetic feature in mammals,in which haploid cells normally arise only as post-meiotic germ cells that serve to ensure a diploid genome upon fertilization. Gamete manipulation has yielded haploid embryonic stem (ES) cells from several mammalian species,but haploid human ES cells have yet to be reported. Here we generated and analysed a collection of human parthenogenetic ES cell lines originating from haploid oocytes,leading to the successful isolation and maintenance of human ES cell lines with a normal haploid karyotype. Haploid human ES cells exhibited typical pluripotent stem cell characteristics,such as self-renewal capacity and a pluripotency-specific molecular signature. Moreover,we demonstrated the utility of these cells as a platform for loss-of-function genetic screening. Although haploid human ES cells resembled their diploid counterparts,they also displayed distinct properties including differential regulation of X chromosome inactivation and of genes involved in oxidative phosphorylation,alongside reduction in absolute gene expression levels and cell size. Surprisingly,we found that a haploid human genome is compatible not only with the undifferentiated pluripotent state,but also with differentiated somatic fates representing all three embryonic germ layers both in vitro and in vivo,despite a persistent dosage imbalance between the autosomes and X chromosome. We expect that haploid human ES cells will provide novel means for studying human functional genomics and development. View Publication

Human embryonic stem cells (hESCs) can be maintained as undifferentiated cells in vitro and induced to differentiate into a variety of somatic cell types. Thus,hESCs provide a source of differentiated cell types that could be used to replace diseased cells of a tissue. The efficient cryopreservation of hESCs is important for establishing effective stem cell banks,however,conventional slow freezing methods usually lead to low rates of recovery after thawing cells and their replating in culture. We have established a method for recovering cryopreserved hESCs using pinacidil and compared it to a method that employs the ROCK inhibitor Y-27632. We show that pinacidil is similar to Y-27632 in promoting survival of hESCs after cryopreservation. The cells exhibited normal hESC morphology,retained a normal karyotype,and expressed characteristic hESC markers (OCT4,SSEA3,SSEA4 and TRA-1-60). Moreover,the cells retained the capacity to differentiate into derivatives of all three embryonic germ layers as demonstrated by differentiation through embryoid body formation. Pinacidil has been used for many years as a vasodilator drug to treat hypertension and its manufacture and traceability are well defined. It is also considerably cheaper than Y-27632. Thus,the use of pinacidil offers an efficient method for recovery of cryopreserved dissociated human ES cells. View Publication

Human embryonic stem cells (hESCs) are self-renewing,pluripotent cells derived from the inner cell mass of blastocysts,early-stage embryos,or blastomeres. hESCs can be propagated indefinitely in an undifferentiated state in vitro and have the ability to differentiate into all cell types of the body. Therefore,these cells can potentially provide an unlimited source of cells and hold promise for transplantation therapy,regenerative medicine,drug screening and discovery,and basic scientific research. Surplus human embryos donated for hESC derivation are extremely valuable,and inefficient derivation of hESCs would be a terrible waste of human embryos. Here,we describe a method for isolating hESC lines from human blastocysts with high efficiency. We also describe the methods for excising differentiated areas from partially differentiated hESC colonies and re-isolating undifferentiated hESCs from extremely differentiated hESC colonies. View Publication

Human somatic cells can be reprogrammed to the pluripotent state to become human-induced pluripotent stem cells (hiPSC). This reprogramming is achieved by activating signaling pathways that are expressed during early development. These pathways can be induced by ectopic expression of four transcription factors—Oct4,Sox2,Klf4,and c-Myc. Although there are many ways to deliver these transcription factors into the somatic cells,this chapter will provide protocols that can be used to generate hiPSC from lentiviruses. View Publication