Extended Data Fig. 4: MICL regulates NET formation.
From: Recognition and control of neutrophil extracellular trap formation by MICL
a, Fluorescence of NET-bound Sytox green of MSU or PMA-induced NETs in thioglycollate-elicited neutrophils analysed using a SPARK CYTO reader (Tecan) every 10 min for up to 7 h. Data is a representative example of n = 3 independent experiments, mean ± SD performed in triplicate. b, Representative Sytox green fluorescent (cyan), Draq5 (magenta), and bright field (grey) images of MSU-induced NETs in bone marrow neutrophils isolated from WT and MICL-deficient animals. Cells were stained unfixed. Quantification (as percentage of Sytox green positive cells extruding NETs) shown as mean ± SD, n = 1 experiment performed in triplicate. Statistical significance determined by Student’s t-test. c, Representative Sytox green fluorescent and bright field images of MSU-induced NETs in thioglycollate-elicited neutrophils isolated from MICL-deficient animals with and without DPI pre-treatment. Scale bar = 100 µm. Quantification (mean ± SD) shown right (n = 2 experiments performed in triplicate). Statistical significance determined by Student’s t-test. d, Fluorescence of NET-bound Sytox of MSU-induced NETs in thioglycollate-elicited neutrophils with and without NSC87877 treatment analysed 4 h after stimulation. Pooled data from two independent experiments performed in duplicate represented as mean ± SD. e, Representative confocal immunofluorescence microscopy images of NETs in CAIA WT and MICL−/− synovial sections on day 11 (as per schedule in Extended Data Fig. 1a). GR-1 (purple), DAPI (blue), Cit-H3 (yellow), and DNA/H1 (green) and quantification of cit-H3/GR-1 area (n = 1 experiment with 3 mice/group). NETs are defined as GR-1+Cit-H3 + DNA/H1+ stained cells. Scale bar = 100 µm. Statistical significance determined by Student’s t-test. f, Representative imaging flow cytometry examples of NET positive (+) and NET negative (−) neutrophils isolated from the ankle joints of mice at day 11 during CAIA. BF, Brightfield; DNA/H1 (green); Ly6G (purple); Cit-H3 (red). *, p < 0.05.
