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

Developing efficacious vaccines against enteric diseases is a global challenge that requires a better understanding of cellular recruitment dynamics at the mucosal surfaces. The current paradigm of T cell homing to the gastrointestinal (GI) tract involves the induction of $$4$$7 and CCR9 by Peyer's patch and mesenteric lymph node (MLN) dendritic cells (DCs) in a retinoic acid-dependent manner. This paradigm,however,cannot be reconciled with reports of GI T cell responses after intranasal (i.n.) delivery of antigens that do not directly target the GI lymphoid tissue. To explore alternative pathways of cellular migration,we have investigated the ability of DCs from mucosal and nonmucosal tissues to recruit lymphocytes to the GI tract. Unexpectedly,we found that lung DCs,like CD103(+) MLN DCs,up-regulate the gut-homing integrin $$4$$7 in vitro and in vivo,and induce T cell migration to the GI tract in vivo. Consistent with a role for this pathway in generating mucosal immune responses,lung DC targeting by i.n. immunization induced protective immunity against enteric challenge with a highly pathogenic strain of Salmonella. The present report demonstrates novel functional evidence of mucosal cross talk mediated by DCs,which has the potential to inform the design of novel vaccines against mucosal pathogens. View Publication

BACKGROUND: Induced pluripotent stem (iPS) cells have the capability to undergo self-renewal and differentiation into all somatic cell types. Since they can be produced through somatic cell reprogramming,which uses a defined set of transcription factors,iPS cells represent important sources of patient-specific cells for clinical applications. However,before these cells can be used in therapeutic designs,it is essential to understand their genetic stability. METHODOLOGY/PRINCIPAL FINDINGS: Here,we describe DNA damage responses in human iPS cells. We observe hypersensitivity to DNA damaging agents resulting in rapid induction of apoptosis after γ-irradiation. Expression of pluripotency factors does not appear to be diminished after irradiation in iPS cells. Following irradiation,iPS cells activate checkpoint signaling,evidenced by phosphorylation of ATM,NBS1,CHEK2,and TP53,localization of ATM to the double strand breaks (DSB),and localization of TP53 to the nucleus of NANOG-positive cells. We demonstrate that iPS cells temporary arrest cell cycle progression in the G(2) phase of the cell cycle,displaying a lack of the G(1)/S cell cycle arrest similar to human embryonic stem (ES) cells. Furthermore,both cell types remove DSB within six hours of γ-irradiation,form RAD51 foci and exhibit sister chromatid exchanges suggesting homologous recombination repair. Finally,we report elevated expression of genes involved in DNA damage signaling,checkpoint function,and repair of various types of DNA lesions in ES and iPS cells relative to their differentiated counterparts. CONCLUSIONS/SIGNIFICANCE: High degrees of similarity in DNA damage responses between ES and iPS cells were found. Even though reprogramming did not alter checkpoint signaling following DNA damage,dramatic changes in cell cycle structure,including a high percentage of cells in the S phase,increased radiosensitivity and loss of DNA damage-induced G(1)/S cell cycle arrest,were observed in stem cells generated by induced pluripotency. View Publication

Characterization of pluripotent stem cells is required for the registration of stem cell lines and allows for an impartial and objective comparison of the results obtained when generating multiple lines. It is therefore crucial to establish specific,fast and reliable protocols to detect the hallmarks of pluripotency. Such protocols should include immunocytochemistry (takes 2 d),identification of the three germ layers in in vitro-derived embryoid bodies by immunocytochemistry (immunodetection takes 3 d) and detection of differentiation markers in in vivo-generated teratomas by immunohistochemistry (differentiation marker detection takes 4 d). Standardization of the immunodetection protocols used ensures minimum variations owing to the source,the animal species,the endogenous fluorescence or the inability to collect large amounts of cells,thereby yielding results as fast as possible without loss of quality. This protocol provides a description of all the immunodetection procedures necessary to characterize mouse and human stem cell lines in different circumstances. View Publication

The generation of endothelial progenitor cells (EPCs) from blood monocytes has been propagated as a novel approach in the diagnosis and treatment of cardiovascular diseases. Low-density lipoprotein (LDL) uptake and lectin binding together with endothelial marker expression are commonly used to define these EPCs. Considerable controversy exists regarding their nature,in particular,because myelomonocytic cells share several properties with endothelial cells (ECs). This study was performed to elucidate whether the commonly used endothelial marker determination is sufficient to distinguish supposed EPCs from monocytes. We measured endothelial,hematopoietic,and progenitor cell marker expression of monocytes before and after angiogenic culture by fluorescence microscopy,flow cytometry,and real-time reverse transcription-polymerase chain reaction. The function of primary monocytes and monocyte-derived supposed EPCs was investigated during vascular network formation and EC colony-forming unit (CFU-EC) development. Monocytes cultured for 4 to 6 days under angiogenic conditions lost CD14/CD45 and displayed a commonly accepted EPC phenotype,including LDL uptake and lectin binding,CD31/CD105/CD144 reactivity,and formation of cord-like structures. Strikingly,primary monocytes already expressed most tested endothelial genes and proteins at even higher levels than their supposed EPC progeny. Neither fresh nor cultured monocytes formed vascular networks,but CFU-EC formation was strictly dependent on monocyte presence. LDL uptake,lectin binding,and CD31/CD105/CD144 expression are inherent features of monocytes,making them phenotypically indistinguishable from putative EPCs. Consequently,monocytes and their progeny can phenotypically mimic EPCs in various experimental models. View Publication

Human embryonic stem cells (hESCs) offer exciting potential in regenerative medicine for the treatment of a host of diseases including cancer,Alzheimer's and Parkinson's disease. They also provide insight into human development and disease and can be used as models for drug discovery and toxicity analyses. The key properties of hESCs that make them so promising for medical use are that they have the ability to self-renew indefinitely in culture and they are pluripotent,which means that they can differentiate into any of more than 200 human cell types. Since proteins are the effectors of cellular processes,it is important to investigate hESC expression at the protein level as well as at the transcript level. In addition,post-translational modifications,such as phosphorylation,may influence the activity of pivotal proteins in hESCs,and this information can only be determined by studying the proteome. In this review,we summarize the results obtained from several proteomics analyses of hESCs that have been reported in the last few years. View Publication

Cryopreservation is the use of low temperatures to preserve structurally intact living cells. The cells that survive the thermodynamic journey from the 37 °C incubator to the -196 °C liquid nitrogen storage tank are free from the influences of time. Thus,cryopreservation is a critical component of cell culture and cell manufacturing protocols. Successful cryopreservation of human cells requires that the cells be derived from patient samples that are collected in a standardized manner,and carefully handled from blood draw through cell isolation. Furthermore,proper equipment must be in place to ensure consistency,reproducibility,and sterility. In addition,the correct choice and amount of cryoprotectant agent must be added at the correct temperature,and a controlled rate of freezing (most commonly 1 °C/min) must be applied prior to a standardized method of cryogenic storage. This appendix describes how human primary cells can be frozen for long-term storage and thawed for growth in a tissue culture vessel. View Publication

We have generated rat monoclonal antibodies specific for the mouse eotaxin receptor,C-C chemokine receptor 3 (CCR3). Several anti-CCR3 mAbs proved to be useful for in vivo depletion of CCR3-expressing cells and immunofluorescent staining. In vivo CCR3 mAbs of the IgG2b isotype substantially depleted blood eosinophil levels in Nippostrongyus brasiliensis-infected mice. Repeated anti-CCR3 mAb treatment in these mice significantly reduced tissue eosinophilia in the lung tissue and bronchoalveolar lavage fluid. Flow cytometry revealed that mCCR3 was expressed on eosinophils but not on stem cells,dendritic cells,or cells from the thymus,lymph node,or spleen of normal mice. Unlike human Th2 cells,mouse Th2 cells did not express detectable levels of CCR3 nor did they give a measurable response to eotaxin. None of the mAbs were antagonists or agonists of CCR3 calcium mobilization. To our knowledge,the antibodies described here are the first mAbs reported to be specific for mouse eosinophils and to be readily applicable for the detection,isolation,and in vivo depletion of eosinophils. View Publication

The translation of laboratory processes into scaled production systems suitable for manufacture is a significant challenge for cell based therapies; in particular there is a lack of analytical methods that are informative and efficient for process control. Here the potential of image analysis as one part of the solution to this issue is explored,using pluripotent stem cell colonies as a valuable and challenging exemplar. The Cell-IQ live cell imaging platform was used to build image libraries of morphological culture attributes such as colony edge� View Publication

Determining how normal and leukemic stem cells behave in vivo,in a dynamic and noninvasive way,remains a major challenge. Most optical tracking technologies rely on the use of fluorescent or bioluminescent reporter genes,which need to be stably expressed in the cells of interest. Because gene transfer in primary leukemia samples represents a major risk to impair their capability to engraft in a xenogenic context,we evaluated the possibility to use gene transfer-free labeling technologies. The lipophilic dye 3,3,3',3' tetramethylindotricarbocyanine iodide (DiR) was selected among 4 near-infrared (NIR) staining technologies. Unfortunately we report here a massive transfer of the dye occurring toward the neighbor cells both in vivo and in vitro. We further demonstrate that all lipophilic dyes tested in this study (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine perchlorate [DiI],DiD,DiR,and PKH26) can give rise to microenvironmental contamination,including when used in suboptimal concentration,after extensive washing procedures and in the absence of phagocytosis or marked cell death. This was observed from all cell types tested. Eventually,we show that this microenvironmental contamination is mediated by both direct cell-cell contacts and diffusible microparticles. We conclude that tracking of labeled cells using non-genetically encoded markers should always be accompanied by drastic cross validation using multimodality approaches. View Publication

Staphylococcal enterotoxins (SE) crosslink the MHC‐II on antigen‐presenting cells (APC) with the T‐cell receptor,inducing a polyclonal T‐cell response. Although APCs are the initial targets of SE and are critical in shaping subsequent T‐cell activation,the effects of SE on APC function remain poorly understood. This study investigates the immunomodulatory effects of staphylococcal enterotoxin A (SEA) on monocytes and their differentiation into monocyte‐derived dendritic cells (moDC) or macrophages (MDM). Transcriptomic analyses of human monocytes via RNA sequencing revealed SEA‐induced enrichment of gene pathways associated with inflammation,infection,and dermatitis,effects that were amplified in the presence of T cells. Phenotypic and functional characterization showed that SEA‐primed monocytes differentiated into MDM with an altered polarization,deviating from classical M1/M2 pathways. SEA‐primed MDM exhibited downregulation of key markers,including HLA‐DR,CD80,CD86,and PD‐L1. Functional assays demonstrated that SEA‐primed MDM pushed hyperinflammatory T‐cell responses,with significantly enhanced proliferation and IFN‐γ secretion. In contrast,following SEA‐priming,moDC retained robust antigen‐presenting capabilities and displayed enhanced expression of molecules involved in T‐cell interactions. These findings provide mechanistic insights into SEA‐mediated immune modulation,illustrating how SEA reprograms MDM functions and amplifies proinflammatory T‐cell responses. This advances our understanding of superantigen‐driven immune interactions,offering a foundation for developing therapeutic strategies to mitigate superantigen‐mediated immune conditions. Staphylococcal enterotoxin A (SEA) alters monocyte differentiation and function,while preserving T cell stimulatory capacity. SEA‐primed macrophages downregulate antigen‐presenting markers yet drive heightened T‐cell proliferation and IFN‐γ secretion. These findings reveal mechanisms of SEA‐mediated immune modulation and superantigen‐driven inflammation. View Publication