Dataset Information

Single-Cell Mass Spectrometry Reveals Changes in Lipid and Metabolite Expression in RAW 264.7 Cells upon Lipopolysaccharide Stimulation.

ABSTRACT: It has been widely recognized that individual cells that exist within a large population of cells, even if they are genetically identical, can have divergent molecular makeups resulting from a variety of factors, including local environmental factors and stochastic processes within each cell. Presently, numerous approaches have been described that permit the resolution of these single-cell expression differences for RNA and protein; however, relatively few techniques exist for the study of lipids and metabolites in this manner. This study presents a methodology for the analysis of metabolite and lipid expression at the level of a single cell through the use of imaging mass spectrometry on a high-performance Fourier transform ion cyclotron resonance mass spectrometer. This report provides a detailed description of the overall experimental approach, including sample preparation as well as the data acquisition and analysis strategy for single cells. Applying this approach to the study of cultured RAW264.7 cells, we demonstrate that this method can be used to study the variation in molecular expression with cell populations and is sensitive to alterations in that expression that occurs upon lipopolysaccharide stimulation. Graphical Abstract.

SUBMITTER: Yang B

PROVIDER: S-EPMC5943162 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Single-Cell Mass Spectrometry Reveals Changes in Lipid and Metabolite Expression in RAW 264.7 Cells upon Lipopolysaccharide Stimulation.

Yang Bo B Patterson Nathan Heath NH Tsui Tina T Caprioli Richard M RM Norris Jeremy L JL

Journal of the American Society for Mass Spectrometry 20180313 5

It has been widely recognized that individual cells that exist within a large population of cells, even if they are genetically identical, can have divergent molecular makeups resulting from a variety of factors, including local environmental factors and stochastic processes within each cell. Presently, numerous approaches have been described that permit the resolution of these single-cell expression differences for RNA and protein; however, relatively few techniques exist for the study of lipid ...[more]

PMID: 29536413

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Project description:Lithium remains the preferred Food and Drug Administration- (FDA-) approved psychiatric drug for treatment of bipolar disorders since its medical establishment more than half a century ago. Recent studies revealed a promising role for lithium in the regulation of inflammation, oxidative stress, and neurodegeneration albeit unclear about its exact mode of action. Thus, the intention of this study is to delineate the regulatory mechanisms of lithium on oxidative stress in lipopolysaccharide- (LPS-) activated macrophages by evaluating its effects on nuclear factor-κB (NF-κB) activity and mRNA expression of multiple oxidative stress-related NF-κB genes. Raw 264.7 macrophages were treated with up to 10 mM lithium, and no change in cell proliferation, viability, growth, and cell adhesion was observed in real time. Pretreatment with low doses of lithium was shown to reduce nitric oxide (NO) production in LPS-activated macrophages. A reduced internal H2DCFDA fluorescence intensity, indicative of reduced reactive oxygen species (ROS) production, was observed in LPS-activated Raw 264.7 macrophages treated with lithium. Lithium has been shown to lower the production of the chemokine RANTES; furthermore, this inhibitory action of lithium has been suggested to be independent of glycogen synthase kinase-3 β (GSK3β) activity. It is shown here that lithium modulates the expression of several inflammatory genes including IκB-α, TRAF3, Tollip, and NF-κB1/p50 which are regulators of the NF-κB pathway. Moreover, lithium inhibits NF-κB activity by lowering nuclear translocation of NF-κB in LPS-activated macrophages. This is the first study to associate Tollip, Traf-3, and IκB-α mRNA expression with lithium effect on NF-κB activity in LPS-activated Raw 264.7 macrophages. Although these effects were obtained using extratherapeutic concentrations of lithium, results of this study provide useful information towards understanding the mode of action of lithium. This study associates lithium with reduced oxidative stress in LPS-activated Raw 264.7 macrophages and further suggests candidate molecular targets for the regulation of oxidative stress-related diseases using lithium beyond bipolar disorders.

Dataset Information

Single-Cell Mass Spectrometry Reveals Changes in Lipid and Metabolite Expression in RAW 264.7 Cells upon Lipopolysaccharide Stimulation.

Publications

Single-Cell Mass Spectrometry Reveals Changes in Lipid and Metabolite Expression in RAW 264.7 Cells upon Lipopolysaccharide Stimulation.

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