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

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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 development and evaluation of a SILAC-labeled primary rat microglia model. Although the isotope labeling scheme for primary microglia is drastically different from that of immortalized cell lines, our de novo and uninterrupted primary culture labeling protocol (DUP-SILAC) resulted in sufficient incorporation of SILAC labels for mass spectrometry-based proteomic profiling. In addition, label incorporation did not alter their morphology and response to endotoxin stimulation. Proteomic analysis of the endotoxin-stimulated SILAC-labeled primary microglia identified expected as well as potentially novel activation markers and pro-inflammatory pathways that could be quantified in a more physiologically relevant cellular model system compared to immortalized cell lines. The establishment of primary microglia SILAC model will further expand our capacity for global scale proteomic profiling of pathways under various physiological and pathological conditions. Proteomic MS data are available via ProteomeXchange with identifier PXD002759.", "claims": [{"id": "claim-target-result", "statement": "The DUP-SILAC strategy established a SILAC-labeled primary rat microglia model with approximately 70% label incorporation, supporting mass-spectrometry proteomic analysis; the raw mass-spectrometry data are available through ProteomeXchange via PRIDE under PXD002759.", "kind": "quantitative", "target": {"value": 70.0, "unit": "per cent", "tolerance": null, "source": "Even though it was not possible to consecutively passage the primary glial cultures, which is the approach used for SILAC labeling of immortalized microglial cell lines, the two-week de novo and uninterrupted primary culture SILAC (DUP-SILAC) labeling strategy achieved a label incorporation ratio of ~70%, a value comparable to that observed for murine primary neuron cultures [20, 21]."}, "evidence": [{"asset": "source:pmid:26936193", "locator": "sections[10] \"p\" paragraph 1", "quote": "Even though it was not possible to consecutively passage the primary glial cultures, which is the approach used for SILAC labeling of immortalized microglial cell lines, the two-week de novo and uninterrupted primary culture SILAC (DUP-SILAC) labeling strategy achieved a label incorporation ratio of ~70%, a value comparable to that observed for murine primary neuron cultures [20, 21]."}, {"asset": "source:pmid:26936193", "locator": "sections[10] \"p\" paragraph 1", "quote": "Furthermore, proteomic analysis revealed that the SILAC labeled primary rat microglia responded in a similar fashion as immortalized rat microglial cells to stimulation with LPS as defined by expression changes of known proteins and related pathways associated with LPS-induced classical activation."}, {"asset": "source:pmid:26936193", "locator": "sections[5] \"p\" paragraph 1", "quote": "The mass spectrometric raw data have been deposited to the ProteomeXchange Consortium [18] via the PRIDE partner repository with the dataset identifier PXD002759."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "partial", "objective": "The DUP-SILAC strategy established a SILAC-labeled primary rat microglia model with approximately 70% label incorporation, supporting mass-spectrometry proteomic analysis; the raw mass-spectrometry data are available through ProteomeXchange via PRIDE under PXD002759.", "steps": [{"id": "read_published_estimate", "order": 1, "name": "Read the published estimate from the source", "kind": "other", "state": "complete", "blocked_on": null, "description": "The claim, method and evidence span published with the source article.", "duration_hours": null, "parameters": {}, "inputs": ["source:pmid:26936193"], "outputs": ["the published estimate and its evidence"], "instruments": [], "reagents": []}, {"id": "obtain_source_data", "order": 2, "name": "Obtain the source data from the named repository", "kind": "data-preparation", "state": "blocked", "blocked_on": "data:accession_absent", "description": "The GEO series accession resolves and its sample accessions are listed. 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