mertk-apc (fab8912a)  (R&D Systems)

Journal: EBioMedicine

Article Title: Pro-inflammatory monocyte profile in patients with major depressive disorder and suicide behaviour and how ketamine induces anti-inflammatory M2 macrophages by NMDAR and mTOR

doi: 10.1016/j.ebiom.2019.10.063

Figure Lengend Snippet: Ketamine induces a M2c-like phenotype in monocyte-derived macrophages with increased levels of MERTK, CD163, and intermediate levels of CD64 while reducing the response to LPS. Monocyte-derived macrophages were differentiated for 7 days in the presence or absence of ketamine (0.1, 1 and 10 µM), and the percentage of (a) MERTK, (b) CD163, (c) CD206 and (d) CD64 positive CD11b + macrophages was analysed by flow cytometry. Macrophage polarization controls were performed using dexamethasone (0.1 µM) for M2c, IL-4 (40 ng/mL) for M2a, and LPS (1 ng/mL) plus IFN-γ (50 ng/mL) for M1. Representative and independent data are shown. (e-i) To analyse the response to an inflammatory stimulus, ketamine-induced macrophages were stimulated for 24h with 1 ng/mL of LPS. The activation markers (e) CD80 and (f) HLADR were evaluated by flow cytometry and (g) TNF-α, (h) IL-6 and (i) IL-10 production was assessed by ELISA. Each dot represents an independent donor and pooled data were graphed. One-way ANOVA test was performed and statistical significance is denoted as * p < 0.05; ** p < 0.01; *** p < 0.001. Untreated condition: Untd; dexamethasone: DEX.

Article Snippet: The phenotype and activation of macrophages were characterized by cell surface staining employing the appropriate combination of directly conjugated antibodies against human CD11b-APC/Cy7 (BioLegend Cat # 101225, RRID: AB_830641), CD64-PE/Cy7 (BioLegend Cat # 305021, RRID: AB_2561583), CD163-PerCP/Cy5.5 (BioLegend Cat # 333625, RRID: AB_2,650629), CD206-AlexaFluor 488 (BioLegend Cat # 321113, RRID: AB_571874), CD14-PE (BioLegend Cat # 325605, RRID: AB_830678), HLA-DR-FITC (BioLegend Cat # 980402, RRID: AB_2616625), CD80-PE (BioLegend Cat # 305207, RRID: AB_314,503), and MERTK-APC (R&D Systems Cat # FAB8912A RRID:AB_357213) along with its control isotype IgG1-APC (R&D Systems, Cat # IC002A).

Techniques: Derivative Assay, Flow Cytometry, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: EBioMedicine

Article Title: Pro-inflammatory monocyte profile in patients with major depressive disorder and suicide behaviour and how ketamine induces anti-inflammatory M2 macrophages by NMDAR and mTOR

doi: 10.1016/j.ebiom.2019.10.063

Figure Lengend Snippet: NMDAR antagonist MK-801, but not the AMPAR antagonist NBQX, induces a similar M2 profile as ketamine, and this phenotype is completely abolished by the inhibition of the mTOR pathway. Monocyte-derived macrophages were differentiated for 7 days in the presence or absence of the NMDAR antagonist MK-801 (1 and 10 µM) or AMPAR antagonist NBQX (1 and 10 µM), and the percentage of (a) MERTK and (b) CD206 was analysed for M2 polarization by flow cytometry. Representative histograms and independent data are shown. Rapamycin (0.01–1 nM), added from day 0, was used to evaluate the role of the mTOR pathway in macrophage polarization after 7 days of culture. Viable CD11b + cells were analysed for the expression of (c) MERTK, (d) CD206, (e) CD64, and (f) CD163. Each experimental condition includes at least 4 independent donors. Pooled data were graphed and one-way ANOVA test was performed accordingly. Statistical significance is denoted as * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The phenotype and activation of macrophages were characterized by cell surface staining employing the appropriate combination of directly conjugated antibodies against human CD11b-APC/Cy7 (BioLegend Cat # 101225, RRID: AB_830641), CD64-PE/Cy7 (BioLegend Cat # 305021, RRID: AB_2561583), CD163-PerCP/Cy5.5 (BioLegend Cat # 333625, RRID: AB_2,650629), CD206-AlexaFluor 488 (BioLegend Cat # 321113, RRID: AB_571874), CD14-PE (BioLegend Cat # 325605, RRID: AB_830678), HLA-DR-FITC (BioLegend Cat # 980402, RRID: AB_2616625), CD80-PE (BioLegend Cat # 305207, RRID: AB_314,503), and MERTK-APC (R&D Systems Cat # FAB8912A RRID:AB_357213) along with its control isotype IgG1-APC (R&D Systems, Cat # IC002A).

Techniques: Inhibition, Derivative Assay, Flow Cytometry, Expressing