Notes

Diverse details as well as internet sites involving activity of cochlear neurotransmitters.
The posttranslational modifications (PTMs) ADP-ribosylation and phosphorylation are important regulators of cellular pathways, and while mass spectrometry (MS)-based methods for the study of protein phosphorylation are well developed, protein ADP-ribosylation methodologies are still in a rapidly developing stage. The method described in this chapter uses immobilized metal affinity chromatography (IMAC), a phosphoenrichment matrix, to enrich ADP-ribosylated peptides which have been cleaved down to their phosphoribose attachment sites by a phosphodiesterase, thus isolating the ADP-ribosylated and phosphorylated proteomes simultaneously. To achieve the robust, relative quantification of PTM-level changes we have incorporated dimethyl labeling, a straightforward and economical choice which can be used on lysate from any cell type, including primary tissue. The entire pipeline has been optimized to work in ADP-ribosylation-compatible buffers and with protease-laden lysate from macrophage cells.Macrophages are professional innate immune cells that are broadly disseminated throughout the body, shape various innate and adaptive immune responses, and play crucial roles in inflammation, homeostasis, wound healing, and tissue remodelling. According to their surrounding microenvironments, macrophages can differentiate themselves in different phenotypes. Over the last two decades, gene expression profiling has been used to decipher new transcripts associated with macrophage phenotypes. This chapter outlines protocols used to isolate and culture murine macrophages and how they can be "polarized" to obtain a specific phenotype. Furthermore, we describe a protocol for gene expression profiling using a quantitative real-time polymerase chain reaction (qPCR), a high-standard technology in the field of gene expression.The co-culture of adipocytes and immune cells, such as macrophages or T cells (CD4+ or CD8+ subsets), is a novel experimental approach used to study paracrine interactions (or the cross talk) between cultured cell types in isolation, in order to understand their role in obese adipose tissue (AT) inflammation and dysfunction. Here we describe the general methodologies required for the co-culture of mature adipocytes (differentiated 3T3-L1 pre-adipocyte cell line) with primary immune cell subsets purified from mouse splenic mononuclear cells using a magnetic MicroBead positive selection, wherein multiple immune cell populations can be purified sequentially from a single mouse spleen, thereby providing diversity in the types of immune cells that can be co-cultured with adipocytes. Additionally, we describe experimental procedures for co-culturing adipocytes and immune cells in two different co-culture systems, including a cell contact-dependent co-culture system, wherein the cells are in direct physical contact, and a cell contact-independent, soluble mediator-driven co-culture system wherein the cells are physically separated by a trans-well semipermeable membrane. Finally, we discuss how these co-culture models can be utilized to recapitulate the AT microenvironment in obesity by utilizing physiologically relevant ratios of adipocytesimmune cells (specifically CDllb+ macrophages, CD4+ T cells, or CD8+ T cells) and lipopolysaccharide stimulation that mimics endotoxin concentrations observed in obesity.Three-dimensional (3D) cultures are better able to reflect the tumor microenvironment than two-dimensional (2D) monolayer cultures by facilitating cell-cell interactions in the appropriate spatial dimensions. Here I describe the isolation and co-culture of immune cells with tumor cell lines in a three-dimensional system facilitated by a basement membrane extract. This allows for further downstream applications to analyze interactions between these cell types.Exosomes are cell-derived vesicles that have been implicated in the pathogenesis of many inflammatory diseases. In the immune system, it has been shown that T lymphocyte-derived exosomes are able to induce diverse cellular responses. There are several methods to isolate and to characterize exosomes, each with their own advantages and disadvantages. Here, we describe a centrifugation approach, combined with mass spectrometry characterization, as a means to study exosomes derived from primary human T lymphocytes. This method is sensitive and therefore can be applied when a limited amount of sample is available.Macrophages play an essential role in diverse biological processes, from the immune response to inflammatory and neurodegenerative disorders, to various cancers. A macrophage subpopulation, known as tumor-associated macrophages (TAMs), has been shown to promote tumorigenesis, metastasis, and immune escape of cancer cells. Some of the pro-tumorigenic effects of TAMs are mediated via the secretion of nano-vesicles (exosomes) from macrophages to neighboring cells. In this chapter, we describe peritoneal macrophage isolation methods, polarization of TAMs, and purification and characterization of macrophage-derived exosomes.Macrophages play a critical role in innate immunity through Toll-like receptor (TLR) signaling. Lipopolysaccharides (LPS) are a ligand of microbial origin that can trigger cell signaling in macrophages through TLRs and production of pro-inflammatory cytokines. Statin, a hypercholesterolemia drug, on the contrary, can reduce inflammatory cytokine production, and inflammation at large. Discovery-based quantitative proteomics is a useful method for unraveling complex protein networks and inter-protein interactions. Blebbistatin in vivo Here, we describe protocols for studying the inflammatory proteomics network in RAW 264.7 cells (a model murine macrophage cell line) with the singular or sequential treatment of LPS and statin. We provide detailed protocols, including a quantitative proteomic analysis by mass spectrometry data, a protein network analysis by bioinformatics, and a validation of target through biochemical methods (e.g., immunocytochemistry, immunoblotting, gene silencing, and real-time PCR).Fatty acids (FAs) are essential for building complex lipids, posttranslational modifications, and energetics. FAs can be imported from extracellular sources or synthesized by cells. The analysis of fatty acid methyl esters (FAMEs) by gas chromatography-mass spectrometry (GC-MS) allows for the quantitative analysis of long-chain and very-long-chain fatty acid content of cells. When coupled with isotopic labeling, this approach can elucidate the synthetic pathways being engaged by the cells, and the relative contribution of synthesis and import to maintain lipid content. Here, we describe a method for total cellular fatty acid analysis in macrophages.
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