Bacterial killing by complement requires membrane attack complex formation via surface‐bound C5 convertases Surface‐bound MAC pores were quantified by.

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Bacterial killing by complement requires membrane attack complex formation via surface‐bound C5 convertases Surface‐bound MAC pores were quantified by flow cytometry or confocal microscopy. AConvertase‐labeled bacteria were exposed to a concentration range of either preassembled C5b6 (C5b6MAC) or a mixture of C5 and C6 (Conv‐MAC), in the presence of 100 nM C7. After washing, 20 nM C8 and 100 nM C9‐Cy3 were added. Controls at 0 nM C5b6 or C5‐C6 confirm that the detected C9‐Cy3 deposition is specifically related to MAC formation (solid lines). Proper insertion of pores was assessed by a previously described shaving method with trypsin (Moskovich & Fishelson, ). Bacteria were first incubated with MAC components for 30 min and subsequently treated with 20 μg/ml trypsin for 15 min at 37°C (dotted lines). BConvertase‐labeled perimCherry/cytoGFP bacteria (Green) exposed to C5b6MAC or Conv‐MAC. Conditions were similar to those in (A); however, C9‐Cy5 was used to detect MAC pores (Red). 100 nM of C5 and C6 or C5b6 was used in combination with 100 nM C7, 20 nM C8, and 100 nM C9‐Cy5. Conv + C5b6MAC and Conv‐MAC images were taken in separate experiments in which laser settings were adjusted to the staining intensity of C9‐Cy5 to properly visualize pore distribution. Scale bars = 3 μm. C, DAtomic force microscopy analysis of E. coli BL21 and MG1655 immobilized using the Poly‐L‐Lysine protocol. (C) Entire bacteria and high‐resolution comparisons of untreated and convertase‐labeled E. coli BL21 exposed to C5‐C9 (Conv‐MAC) for 10 min. Scale bars: 800 nm (left) and 30 nm (right). Height scales: 1 μm (left), 8 nm (top right), 22 nm (bottom right). Width of magnification boxes: 42 nm, height scales: 8 nm (top) and 13 nm (bottom). Arrows highlight E. coli porin structures; an asterisk highlights the C5b‐7 stalk. Height profiles (bottom) are shown for the white dashed lines in the images. (D) Atomic force microscopy (height and phase images) of convertase‐labeled E. coli MG1655 exposed to a mixture of C5 and C6 (Conv‐MAC) or preassembled C5b6 (Conv + C5b6MAC), in the presence of C7, C8, C9, FB, and FD. Images were generated in the same experiment. Scale bars: 50 nm. Height scales: 15 nm. This figure and three other replicates are included in Fig EV5B and C. Data information: (A) Data represent mean ± SD of 3 independent experiments. 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: 4, First published: 14 January 2019, DOI: (10.15252/embj.201899852)