Extended Data Fig. 1: PS externalization during NETs formation and the generation of XKR8-deficient mice.
a, Quantification of NETs based on DAPI fluorescence intensity and area. Cells with nuclear area greater than 80 µm2 were classified as NETs. b, PS exposure and NETs formation in human peripheral blood neutrophils (PMNs) from healthy donors treated with PMA (100 nM) at indicated time points, assessed by flow cytometry and fluorescence microscopy. Percentage of Annexin V+ DAPI− cells (representing PS externalization) and NETs per field (quantified by ImageJ software, and an average of ten different fields per donor) are shown. Each dot represents an individual donor (n = 8). c, d, As in b, PS exposure and NETs formation in ATRA-differentiated neutrophil-like HL-60 cells (dHL-60) treated with PMA (100 nM) for 1-4 h. Annexin V staining in live cells (PI−) assessed by flow cytometry. Representative histograms of Annexin V staining (c) and quantifications of Annexin V+PI− cells (n = 3) and NETs (n = 8) (d). e, f, Normalized XKR8 gene expression in human (e) or mouse (f) immune cell subsets, analyzed using data from the Human Protein Atlas (HPA) and Immunological Genome Project (ImmGen), respectively. NETs were defined by a nuclear area greater than 80 µm2 in human PMNs (b) or 100 µm2 in dHL-60 cells (d). Each dot represents an individual donor (b) or a biological replicate (d). Data are represented as mean ± SEM from one representative experiment (d). All experiments were performed for at least two independent times.
