Dataset Information

Development and evaluation of a SILAC method for proteomic analysis of rat primary microglia

ABSTRACT: Microglia play important and dynamic roles in mediating a variety of physiological and pathological processes during the development, normal function, and degeneration of the central nervous system. We report here the development of a SILAC-labeled model system suitable for mass spectrometry-based proteomic profiling in primary rat microglia. A mixed primary glia culture was first established by isolating newborn brain cells from Sprague-Dawley rats and maintaining the culture in a fetal bovine serum-supplemented and chemically defined media containing either the regular amino acids (light label media) or stable isotope-labeled amino acids (heavy label media). After isolation of microglia from the mixed glia cultures, mass spectrometric analysis indicated that the incorporation efficiency of the heavy-labeled amino acids was between 70-80%. Upon stimulation with bacterial endotoxin lipopolysaccharide to induce classical (M1) activation, the light- and heavy-labeled primary microglia responded indistinguishably as judged by the up-regulation of inducible nitric oxide synthase. More important, the relative quantitation accuracy and proteome coverage obtained by the reported SILAC model allowed for identification of classical microglial activation markers and pathways involved in the proinflammatory response of microglia as determined by bioinformatic analysis. The establishment of this viable primary microglia SILAC model will further expand our capacity for global scale proteomic profiling of pathways mediating the multifaceted microglial responses in various physiological and pathological processes.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Primary Cell, Microglial Cell

DISEASE(S): Disease Free

SUBMITTER: Ashley Cochran

LAB HEAD: Stanley M. Stevens, Jr.

PROVIDER: PXD002759 | Pride | 2016-04-07

REPOSITORIES: Pride

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	1 from 2014.10.03 Primary Microglia Labeling Efficiency_ AC_Mg_week1_lab_eff_100214 and 2 more.mzid_1__from__2014.10.03_Primary_Microglia_Labeling_Efficiency__AC_Mg_week1_lab_eff_100214_and_2_more.pride.mgf.gz	Mzid
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Publications

Characterization of a SILAC method for proteomic analysis of primary rat microglia.

Zhang Ping P Culver-Cochran Ashley E AE Stevens Stanley M SM Liu Bin B

Proteomics 20160413 9

Microglia play important and dynamic roles in mediating a variety of physiological and pathological processes during the development, normal function and degeneration of the central nervous system. Application of SILAC-based proteomic analysis would greatly facilitate the identification of cellular pathways regulating the multifaceted phenotypes of microglia. We and others have successfully SILAC-labeled immortalized murine microglial cell lines in previous studies. In this study, we report the ...[more]

PMID: 26936193

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