GPVI critically contributes to local neutrophil activation in response to LPS. (A) Representative confocal micrographs of neutrophils (Ly6G⁺, yellow), platelets (GPIX⁺, cyan), vessels (CD31⁺, magenta), and DAPI (nucleus, blue) exemplifying PNC cluster in immunofluorescence-stained cryosections of the lung (left) and during intravital confocal microscopy (right). The dashed line marks the region of the magnified excerpt on the right without the vessel channel. Detailed representative images of immunofluorescence-stained cryosections were acquired with a TCS-SP8 confocal laser scanning microscope (Leica Microsystems) with a 63× objective (HC PL APO CS2 63×/1.40 OIL, Leica Microsystems). Intravital confocal microscopy was performed with a TCS-SP8 confocal laser scanning microscope (Leica Microsystems) with a 25× objective (HC FLUOTAR L 25×/0.95 WATER, Leica Microsystems) after 1 hour of LPS treatment and mice received fluorescently labeled antibody derivates to label neutrophils, platelets, and vessels. Representative images derived from videos of the time interval 40 to 45 minutes showing accumulation of neutrophils as cluster with incorporated platelets. Scale bar, 30 μm. (B) Quantification of PNC clusters by manual counting in lung cryosections. Each data point represents the mean number of PNC clusters in 30 FOV for 1 mouse. (C) Fraction of neutrophils that formed PNC clusters. (D) Representative confocal micrographs of neutrophils (Ly6G⁺, yellow), platelets (GPIX⁺, cyan), NETs (Hiscit3⁺, red), and DAPI (nucleus, blue) exemplifying NET-forming neutrophils in immunofluorescence-stained cryosections of the lung. The dashed line marks the region of the magnified excerpt on the right without the vessel channel. Detailed representative images of immunofluorescence-stained cryosections were acquired with a TCS-SP8 confocal laser scanning microscope (Leica Microsystems) with a 63× objective (HC PL APO CS2 63×/1.40 OIL, Leica Microsystems). Scale bar, 30 μm. (E) Quantification of NET-forming neutrophils by manual counting in lung cryosections. Each data point represents the mean number of NET-forming neutrophils in 30 FOV for 1 mouse. (F) Fraction of neutrophils that formed NETs. Each data point represents mean value for 1 mouse. Bars represent mean per group ± standard deviation. The two-way ANOVA with a Šidák multiple comparisons test. ∗P ≤ .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ND, not detectable.