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

Monoclonal antibodies (mAbs) have proven to be instrumental in the advancement of research,diagnostic,industrial vaccine,and therapeutic applications. The use of mAbs in laboratory protocols has been growing in an exponential fashion for the last four decades. Described herein are methods for the development of highly specific mAbs through traditional hybridoma fusion. For ultimate success,a series of simultaneously initiated protocols are to be undertaken with careful attention to cell health of both the myeloma fusion partner and immune splenocytes. Coordination and attention to detail will enable a researcher with basic tissue culture skills to generate mAbs from immunized rodents to a variety of antigens (including proteins,carbohydrates,DNA,and haptens) (see Note 1). Furthermore,in vivo and in vitro methods used for antigen sensitization of splenocytes prior to somatic fusion are described herein. View Publication

The anisotropic alignment of cardiomyocytes in native myocardium tissue is a functional feature that is absent in traditional in vitro cardiac cell culture. Microenvironmental factors cue structural organization of the myocardium,which promotes the mechanical contractile properties and electrophysiological patterns seen in mature cardiomyocytes. Current nano- and microfabrication techniques,such as photolithography,generate simplified cell culture topographies that are not truly representative of the multifaceted and multi-scale fibrils of the cardiac extracellular matrix. In addition,such technologies are costly and require a clean room for fabrication. This chapter offers an easy,fast,robust,and inexpensive fabrication of biomimetic multi-scale wrinkled surfaces through the process of plasma treating and shrinking prestressed thermoplastic. Additionally,this chapter includes techniques for culturing stem cells and their cardiac derivatives on these substrates. Importantly,this wrinkled cell culture platform is compatible with both fluorescence and bright-field imaging; real-time physiological monitoring of CM action potential propagation and contraction properties can elucidate cardiotoxicity drug effects. View Publication

Human sensory neurons are inaccessible for functional examination,and thus little is known about the mechanisms mediating touch sensation in humans. Here we demonstrate that the mechanosensitivity of human embryonic stem (hES) cell-derived touch receptors depends on PIEZO2. To recapitulate sensory neuron development in vitro,we established a multistep differentiation protocol and generated sensory neurons via the intermediate production of neural crest cells derived from hES cells or human induced pluripotent stem (hiPS) cells. The generated neurons express a distinct set of touch receptor-specific genes and convert mechanical stimuli into electrical signals,their most salient characteristic in vivo. Strikingly,mechanosensitivity is lost after CRISPR/Cas9-mediated PIEZO2 gene deletion. Our work establishes a model system that resembles human touch receptors,which may facilitate mechanistic analysis of other sensory subtypes and provide insight into developmental programs underlying sensory neuron diversity. View Publication

Biochemical and biophysical stimuli of stem cell niches finely regulate the self-renewal/differentiation equilibrium. Replicating this in vitro is technically challenging,making the control of stem cell functions difficult. Cell derived matrices capture certain aspect of niches that influence fate decisions. Here,aligned fibrous matrices synthesized by MC3T3 cells were produced and the role of matrix orientation and stiffness on the maintenance of stem cell characteristics and adipo- or osteo-genic differentiation of murine mesenchymal stem cells (mMSCs) was investigated. Decellularized matrices promoted mMSC proliferation. Fibrillar alignment and matrix stiffness work in concert in defining cell fate. Soft matrices preserve stemness,whereas stiff ones,in presence of biochemical supplements,promptly induce differentiation. Matrix alignment impacts the homogeneity of the cell population,that is,soft aligned matrices ameliorate the spontaneous adipogenic differentiation,whereas stiff aligned matrices reduce cross-differentiation. We infer that mechanical signaling is a dominant factor in mMSC fate decision and the matrix alignment contributes to produce a more homogeneous environment,which results in a uniform response of cells to biophysical environment. Matrix thus produced can be obtained in vitro in a facile and consistent manner and can be used for homogeneous stem cell amplification or for mechanotransduction-related studies. View Publication

This protocol offers an ex vivo method for screening host-targeting antivirals (HTAs) using human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs). Unlike virus-targeting antivirals (VTAs),HTAs provide advantages in overcoming drug resistance and offering broad-spectrum protection,especially against rapidly mutating or newly emerging viruses. By focusing on PBMCs or pDCs,known for their high production of humoral factors such as Type I interferons (IFNs),the protocol enables the screening of antivirals that modulate immune responses against viruses. Targeting host pathways,especially innate immunity,allows for species-independent antiviral activity,reducing the likelihood of viral escape mutations. Additionally,the protocol's versatility makes it a powerful tool for testing potential antivirals against various viral pathogens,including emerging viruses,positioning it as an essential resource in both pandemic preparedness and broad-spectrum antiviral research. This approach differentiates itself from existing protocols by focusing on host immune modulation through pDCs,offering a novel avenue for HTA discovery. Key features • Optimized protocol for screening HTAs against dengue virus (DENV),chikungunya virus (CHIKV),and Zika virus (ZIKV). • This protocol is ideal for screening soluble or intravenous-formulated compounds for evaluating their efficacy in experimental settings. • This protocol builds upon the method developed by Tsuji et al. [1] and extends its application to PBMCs and testing against DENV,CHIKV,and ZIKV. View Publication

DNA methylation plays an important role in various biological processes,including cell differentiation,ageing,and cancer development. The most important methylation in mammals is 5-methylcytosine mostly occurring in the context of CpG dinucleotides. Sequencing methods such as whole-genome bisulfite sequencing successfully detect 5-methylcytosine DNA modifications. However,they suffer from the serious drawbacks of short read lengths and might introduce an amplification bias. Here we present Rockfish,a deep learning algorithm that significantly improves read-level 5-methylcytosine detection by using Nanopore sequencing. Rockfish is compared with other methods based on Nanopore sequencing on R9.4.1 and R10.4.1 datasets. There is an increase in the single-base accuracy and the F1 measure of up to 5 percentage points on R.9.4.1 datasets,and up to 0.82 percentage points on R10.4.1 datasets. Moreover,Rockfish shows a high correlation with whole-genome bisulfite sequencing,requires lower read depth,and achieves higher confidence in biologically important regions such as CpG-rich promoters while being computationally efficient. Its superior performance in human and mouse samples highlights its versatility for studying 5-methylcytosine methylation across varied organisms and diseases. Finally,its adaptable architecture ensures compatibility with new versions of pores and chemistry as well as modification types. Subject terms: Genome informatics,Epigenomics,Computational models,DNA sequencing,DNA methylation View Publication

During the metastatic cascade,cancer cells travel through the bloodstream as circulating tumor cells (CTCs) to a secondary site. Clustered CTCs have greater shear stress and treatment resistance,yet their biology remains poorly understood. We therefore engineered a tunable superhydrophobic array device (SHArD). The SHArD-C was applied to culture a clinically relevant model of CTC clusters. Using our device,we cultured a model of cancer cell aggregates of various sizes with immortalized cancer cell lines. These exhibited higher E-cadherin expression and are significantly more capable of surviving high fluid shear stress-related forces compared to single cells and model clusters grown using the control method,helping to explain why clustering may provide a metastatic advantage. Additionally,the SHArD-S,when compared with the AggreWell 800 method,provides a more consistent spheroid-forming device culturing reproducible sizes of spheroids for multiple cancer cell lines. Overall,we designed,fabricated,and validated an easily tunable engineered device which grows physiologically relevant three-dimensional (3D) cancer models containing tens to thousands of cells. View Publication