Fig 1: Ingestion of dual IgM– and C3b/iC3b–opsonized human red blood cells by Fcer1g/Tyrobp double KO (dKO) macrophages. Ingestion of dual IgM– and complement C3b/iC3b–opsonized human red blood cells (hRBCs) by Fcer1g/Tyrobp dKO macrophages (M?s), which lack the ITAM-containing Fc receptor ? chain and the ITAM adaptor DAP12. Time-lapse 3D imaging for 30 min was performed by spinning disk confocal microscopy. Macrophages were labeled green fluorescent with Alexa Fluor 488–conjugated anti-F4/80 antibodies, and the cytosol of hRBCs was loaded with the red fluorescent probe pHrodo Red. Scale bars, 10 µm. IgM, immunoglobulin M; ITAM, immunoreceptor tyrosine-based activation motif.
Fig 2: Slow sinking phagocytosis mediated by complement receptors in Syk conditional KO (cKO) macrophages presented with dual IgM– and complement C3b/iC3b–opsonized targets.A and B, partial or complete ingestion of dual IgM– and complement C3b/iC3b–opsonized human red blood cells (hRBCs) by myeloid-restricted Syk conditional KO (cKO) macrophages. The inset in the lower panel (B) shows an XY optical section. 3D time-lapse imaging was performed by spinning disk confocal microscopy. Macrophages were labeled green fluorescent with Alexa Fluor 488–conjugated anti-F4/80 antibodies, and the cytosol of hRBCs was loaded with the red fluorescent probe pHrodo Red. Scale bars, 10 µm. C, cumulative plots of phagocytic (ingestion) events for WT and Syk cKO macrophages presented with dual IgM– and complement C3b/iC3b–opsonized hRBCs. The x-axes show individual macrophages. D, summary box plots of phagocytic efficiency, defined as the percentage of dual IgM– and complement C3b/iC3b–opsonized hRBCs (in contact with a macrophage), which were fully ingested. Medians are represented by a horizontal line with a lilac-colored circle. Data were analyzed using the Mann–Whitney U test (U = 1002, p =0.6); n (number of macrophages) = 31 (from 3 WT mice) and n = 61 (from 3 Syk cKO mice). n.s. = not significant (p > 0.05). IgM, immunoglobulin M.
Fig 3: Decreased tissue infiltration and altered polarization of muscle-derived F4/80+ cells in the absence of RetSat. (A) The number of CD45+ leukocytes per injured muscle and (B) MCP-1 mRNA expression levels of muscle-derived CD45+ leukocytes from wild-type or RetSat-null mice, determined by qRT-PCR following CTX-induced injury (n = 4). (C) Ratio of anti-F4/80 antibody-stained M?s and anti-Ly6G/Ly6C (GR-1)-stained neutrophils within the CD45+ leukocyte populations in the TA muscles from RetSat+/+ and RetSat-/- mice during the first 4 days of regeneration following CTX-induced injury (n = 3). (D) Expression of MFG-E8 mRNA and (E) of NPY mRNA in muscle-derived CD45+ leukocytes, in total TA muscles, and various organs of RetSat+/+ and RetSat-/- mice, determined by qRT-PCR following CTX-induced injury (n = 4). (F) Representative scatter plots of CD206- and Ly6C-stained muscle-derived F4/80+ cells and the percentages of Ly6C+, CD206+, and MHCIIhigh cells within the muscle-derived F4/80+ population determined at the indicated days following CTX-induced injury in the TA muscles of RetSat+/+ and RetSat-/- mice (n = 3). Asterisks indicate statistical significance (* p < 0.05, ** p < 0.01); ns, not significant.
Fig 4: ErbB4 is induced during DSS colitis and is expressed on Ly6C+ macrophages. (a) Colonic mucosa from mice subjected to DSS colitis were analyzed by flow cytometry to determine percent of cells that are ErbB4+ in colons from mice receiving no DSS (WATER), after 4 days of 3% DSS (INJURY), and after 4 days 3% DSS followed by 3 days without DSS (INFLAM). (b) Analysis of F4/80+/CD11b+ macrophages as a proportion of total mucosal cellularity at indicated time points. (c) Ly6C+/ErbB4+ and Ly6C-/ErbB4+ populations were analyzed. For (a–c), n=9–10 mice per group from three independent DSS colitis experiments. (d) Colonic mucosa from control (WATER) and DSS inflammatory phase (INFLAM) mice were subjected to flow cytometric analysis for Ly6G (neutrophil marker) and ErbB4. Percentage of Ly6G+ cells expressing detectable ErbB4 is shown (n=5 mice per condition). Error bars represent S.E.M. *P<0.05; **P<0.01; ***P<0.001
Fig 5: Slow sinking phagocytosis in macrophages lacking the ITAM adaptor Fc receptor ? chain and DAP12.A, slow partial sinking of dual IgG– and complement C3b/iC3b–opsonized human red blood cells (hRBCs) into Fcer1g/Tyrobp double KO (dKO) macrophages (M?s), which lack the ITAM adaptor proteins Fc receptor ? chain and DAP12. Time-lapse 3D imaging for 16 min was performed by spinning disk confocal microscopy. Macrophages were labeled green fluorescent with Alexa Fluor 488–conjugated anti-F4/80 antibodies, and the plasma membrane was labeled red fluorescent with CellMask Orange. Scale bars, 10 µm. B, cumulative plots of phagocytic (ingestion) events for WT and Fcer1g/Tyrobp dKO macrophages presented with dual IgG– and complement C3b/iC3b–opsonized hRBCs. The x-axes show individual macrophages. C, summary box plots of phagocytic efficiency, defined as the percentage of dual IgG– and complement C3b/iC3b–opsonized hRBCs (in contact with a macrophage) which were fully ingested. Medians are represented by a horizontal line with a lilac-colored circle. Data were analyzed using the Mann–Whitney U test (U = 1465.5, p < 0.0001); n (number of macrophages) = 46 (from 2 WT mice) and n = 35 (from 2 Fcer1g/Tyrobp dKO mice). ***p < 0.0001. IgG, immunoglobulin G; ITAM, immunoreceptor tyrosine-based activation motif.
Supplier Page from Thermo Fisher Scientific for F4/80 Antibody Alexa Fluor 488