Dataset Information

Quantification of O-GlcNAc protein modification in neutrophils by flow cytometry.

ABSTRACT: Observations of intracellular O-linked beta-N-acetylglucosamine (O-GlcNAc) protein modification are primarily performed by Western blot or immunofluorescence microscopy. The goal of this study was to develop a flow cytometric-based assay for O-GlcNAc signaling and thus provide a more quantitative and rapid method to facilitate clinical analyses. Isolated peripheral blood neutrophils were stimulated with fMLF after adherence to glass cover slips. Cells in suspension were treated with either fMLF or PMA. Unstimulated cells served as controls. Neutrophils were fixed with formaldehyde and permeabilized with cold methanol before intracellular O-GlcNAc staining. Cells on cover slips were analyzed by fluorescence microscopy, and suspension cell data were acquired by flow cytometry. O-GlcNAc protein modification was increased following neutrophil stimulation with either 100 nM fMLF or 10 nM PMA. Increases were detected following either treatment using both flow cytometry and fluorescence microscopy. The time necessary for the completion of staining, data acquisition, and analysis was considerably less using flow cytometry. In addition, flow cytometry allows for the analysis of a substantially greater number of cells. Neutrophil protein modifications by O-GlcNAc are rapidly detected using flow cytometry and provide information similar to that observed using fluorescence microscopy.

SUBMITTER: Madsen-Bouterse SA

PROVIDER: S-EPMC3554246 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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Quantification of O-GlcNAc protein modification in neutrophils by flow cytometry.

Madsen-Bouterse Sally A SA Xu Yi Y Petty Howard R HR Romero Roberto R

Cytometry. Part A : the journal of the International Society for Analytical Cytology 20080701 7

Observations of intracellular O-linked beta-N-acetylglucosamine (O-GlcNAc) protein modification are primarily performed by Western blot or immunofluorescence microscopy. The goal of this study was to develop a flow cytometric-based assay for O-GlcNAc signaling and thus provide a more quantitative and rapid method to facilitate clinical analyses. Isolated peripheral blood neutrophils were stimulated with fMLF after adherence to glass cover slips. Cells in suspension were treated with either fMLF ...[more]

PMID: 18524014

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Project description:Mitochondria are highly dynamic organelles whose fusion and fission play an increasingly important role in a number of both normal and pathological cellular functions. Despite the increased interest in mitochondrial dynamics, robust, and quantitative methods to analyze mitochondrial fusion and fission activity in intact cells have not been developed. The current state-of-the art method to measure mitochondrial fusion activity is the polyethylene glycol (PEG) fusion assay in which cells expressing distinct mitochondrially-targeted fluorescent proteins (FPs) are fused together and mitochondrial fusion activity is determined by the rate at which color mixing occurs. Although this assay is useful, cell-cell fusion events are rare, and finding the number of fused cells required to generate statistically rigorous data is both tedious and time-consuming. Furthermore, the data-collection methods available for fluorescence microscopy lead to inherent selection biases that are difficult to control for. To that end, we have developed an unbiased and high-throughput method to detect, image, and analyze fused cells using the Amnis ImagestreamX™ MKII. With IDEAS™ software, we developed algorithms for identifying the fused cells (two nuclei within a single cell), distinguishing them from cell aggregates. Additionally, using the fluorescence localization of the mitochondrially-targeted fluorescent proteins (YFP and DsRed), we applied a modified co-localization algorithm to identify those cells that had a high co-localization score indicating mitochondrial fusion activity. These algorithms were tested using negative controls (FPs associated with fusion deficient mitochondria) and positive controls (cells expressing both FPs in the same mitochondria). Once validated these algorithms could be applied to test samples to evaluate the degree of mitochondrial fusion in cells with various genetic mutations. Ultimately, this new method is the first robust, high-throughput way to directly measure mitochondrial fusion in intact cells. Given how many cellular processes are being linked mitochondrial dynamics, this technique will provide a powerful new tool in the study of this important organelle. © 2016 International Society for Advancement of Cytometry.

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Dataset Information

Quantification of O-GlcNAc protein modification in neutrophils by flow cytometry.

Publications

Quantification of O-GlcNAc protein modification in neutrophils by flow cytometry.

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