APhagocytosis assay of apoptotic or necrotic GFP‐expressing RAW264

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Triggering MSR1 promotes JNK‐mediated inflammation in IL‐4‐activated macrophages APhagocytosis assay of apoptotic or necrotic GFP‐expressing RAW264.7 cells in M2 (IL‐4 or IL13) and untreated M0 BMDMs. BPhagocytosis of fluorescent negatively charged carboxylated and positively charged amino microspheres (B) in primary M2 (IL‐4) and M0 macrophages. Cytochalasin D (Cyto) (6 μM) was used as an inhibitor of phagocytosis, 1 h before phagocytosis. C–EReal‐time fluorescence assays for intraphagosomal proteolysis (C), acidification (D) and lipolysis (E) show substantially increased proteolysis, acidification and lipolysis in the phagosomes of M2 (IL‐4) macrophages. The kinetics of proteolysis, acidification and lipolysis of phagocytosed beads were plotted as a ratio of substrate fluorescence to calibration fluorescence. Beads were added to macrophages at 0 min. (E) is a representative of three independent experiments. Leupeptin (100 nM) and bafilomycin (100 nM) treatments serve as negative controls in (C) and (D), respectively. Data information: The statistical significance of data is denoted on graphs by asterisks where *P < 0.05, ***P < 0.001 or ns = not significant. (A, B) Data are shown as means of relative fluorescence units (RFU) ± standard error of the mean (SEM), Student's t‐test used. (C, D) Shaded area represents SEM. (A–D) Three replicates were used. IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. EMBO J, Volume: 38, Issue: 11, First published: 26 April 2019, DOI: (10.15252/embj.2018100299)