Journal: The Journal of Immunology Author Choice
Article Title: Identification of novel protein biomarkers of macrophage polarization using comparative proteomic analyses of murine primary macrophages
doi: 10.1093/jimmun/vkaf202
Figure Lengend Snippet: Murine primary M0 macrophages were polarized into M1 or M2 macrophages. M0 BMDMs were incubated with LPS for 8 h to elicit M1 polarization or incubated with mouse IL-4 for 24 h to elicit M2 polarization. The cells were collected and subjected to polarization validation using qPCR (A and B), immunoblot (C), and flow cytometry (D). (A) Transcript levels of the well-established M1 biomarkers in M0, M1, and M2 macrophages. (B) Transcript levels of the well-established M2 biomarkers in M0, M1, and M2 macrophages. (C) Immunoblot analysis validated the unique expression of M1 biomarker Ccl5 in M1 cells and of M2 biomarker Clec7a in M2 macrophages. Gapdh served as a loading control. (D) Flow cytometry using antibodies against M1 biomarker iNOS and M2 biomarker Arg1 validated that the majority of LPS-treated macrophages were iNOS positive and IL-4-treated macrophages were mainly Arg1 positive. Data in the bar graphs are presented as mean ± SEM. n = 3–4. ** P < 0.01 relative to M0 BMDMs.
Article Snippet: Primary antibodies used in this study included antibodies against glyceraldehyde-3-phosphate dehydrogenase (Gapdh; Cell Signaling, Boston, MA, USA, 2118; 1:1,000); C-C motif chemokine ligand 5 (Ccl5; Proteintech, Rosemont, IL, USA, 12000-1-AP; 1:500); mouse C-type lectin domain family 4, member e (Clec4e; Novus Biologicals, Centennial, CO, USA, NBP2-27093SS; 1:250); human CLEC4E (Proteintech, 19376-1-AP; 1:1,000); C-type lectin domain family 7, member a (Clec7a; R&D Systems, Minneapolis, MN, USA, AF1756-SP; 1:2,000); mouse CD72 antigen (Cd72; R&D Systems, AF1279SP; 1:1,000); guanylate binding protein 2 (Gbp2; Proteintech, 11854-1-AP; 1:1,000); mouse aconitate decarboxylase 1 (Acod1; Cell Signaling, 19857T; 1:1,000); human ACOD1 (Cell Signaling, 77510T; 1:1,000); and neurochondrin (Ncdn; Proteintech, 13187-1-AP; 1:1000).
Techniques: Incubation, Biomarker Discovery, Western Blot, Flow Cytometry, Expressing, Control
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