Fig. 6: EGFR deletion in myeloid cells increased kidney macrophage efferocytosis ability.
Kidney macrophages were isolated with F4/80 microbeads from myeloid EGFR−/− mice and WT mice 2 days after bilateral 35-min renal pedicle clamping. Bone marrow neutrophils were isolated from WT mice with Ly6G microbeads. Myeloid EGFR−/− mouse kidney macrophages had increased ability to clear apoptotic neutrophils as indicated by: A Ex vivo pHrodo Red real-time efferocytosis assay with apoptotic bone marrow neutrophils (n = 6). B The full gating strategies for the flow cytometry to evaluate kidney macrophage efferocytosis. The effector kidney macrophages were isolated from WT, myeloid EGFR−/− and NeutEGFR−/− mice 2 days after ischemic injury using anti-F4/80 microbeads and stained with CytoTellTM Blue dye. The bait bone marrow neutrophils were isolated from WT mice using anti-ly6G microbeads and stained with CFSE dye and apoptosis was induced by staurosporine. The effector cells alone and bait cells alone or a mixture of effectors and baits at a ratio of 1:4 were incubated overnight before processing for flow cytometry. For flow cytometry analysis strategy, first small debris were removed, and singlets selected. Cells were gated on CytoTellTMblue positivity and CFSE positivity with macrophages only in the reaction or neutrophils only in reaction as controls. When both CFSE-labeled apoptotic neutrophils and CytoTellTMblue-labeled macrophages were both in the reaction, CFSE and CytoTellTMblue double positivity represented macrophages with engulfed apoptotic neutrophils (expressed as percentage of total events with event counts in parenthesis). C EGFR deletion in myeloid cells increased kidney macrophage efferocytosis ability (n = 4 and 6). Data are means ± SEM, **P < 0.01, ***P < 0.001, analyzed using 2 tailed Student’s t test.
