Colocalization of FX in human macrophages. (A-E) Widefield microscopy images of Duolink-PLA assay between FX and MMR (A), DC-SIGN (B), CLEC10A (C), or SR-AI (D) in THP1-derived macrophages, or between FX and SR-AI in CD14⁺-derived macrophages (E) incubated with either PBS (top panels) or FX (bottom panels). Nuclei were counterstained using DAPI. Bars represent 10 µm; objective, ×40. Red spots indicate a distance <40 nm between 2 antigens. Images are representative of 3 different experiments. (F-I) Confocal analysis of immunofluorescent staining for FX (red) and MMR (F) or SR-AI (G) in a THP1-derived macrophage incubated with FX. Cell stack was reconstituted in orthoview to visualize colocalization of the 2 signals (right panels). Arrows indicate areas of colocalization. Z depth is 0.5 µm; bars represent 10 µm; objective, ×63. The mean FX fluorescence of the cells was quantified using Fiji software (H). tMC represents a statistical parameter verifying whether fluorescent signals truly overlap and was calculated using JACoP plugin in Fiji for THP1-derived macrophages incubated with FX and immunostained for FX and MMR (negative control) or SR-AI (I). ***P < .001, respectively, in the Mann-Whitney nonparametric unpaired statistical test. Dots represent each individual cell value and bars represent the mean ± SD of 7 (FX/MMR) to 12 (FX/SR-AI) cells from 3 independent experiments. PBS, phosphate-buffered saline.