Figure 2.
Platelet and neutrophil interactions observed in response to immune complexes. (A-C) Schematic representations (left panels) of different cellular interactions recorded in the brain of both FcγRIIATGN and FcγRIIAnull (Ly6gCre+/−::Rosa26-TdT+/−::CD41-YFP+/−) mice injected with immune complexes and observed with a high-speed widefield fluorescence microscope (right panels). (A) Platelet (cyan) and neutrophil (red) interactions can be associated with partial adhesion (rolling) of neutrophils (neutrophil 2) on blood vessels. (B) “Kiss and fly interaction,” in which an adhered neutrophil (red) briefly interacts with circulating platelets (cyan). (C) Neutrophils (red) adhere to blood vessels in response to immune complexes. Circulating platelets (cyan) interact (white arrows) with both immobilized and rolling neutrophils (yellow arrows). Bars represent 50 µm. The direction of blood flow is indicated with double white arrow heads. Time following the injection of immune complexes is indicated in the inferior right corner (00:00 = minutes:seconds or 00:00:00 = hours:minutes:seconds). Rolling and adhered neutrophils are indicated with yellow arrows, and neutrophil–platelet interactions are indicated with white arrows. (D) Schematic representation of the computer analysis used to track neutrophil trajectories and speed: (1) Image stabilization using Mathworks Matlab 2018b; (2) Neutrophil tracking performed with Bitplane Imaris 7.6 software; (3-4) Automated classification of trajectories using a program in Mathworks Matlab 2018b. (E) Recorded neutrophil trajectories (white lines) in the blood vessels of FcγRIIAnull and FcγRIIATGN mice injected with diluent or immune complexes (Bitplane Imaris 7.6 software). Neutrophils are color coded according to their speed: from dark blue (0 µm/sec) to dark red (4000 µm/sec). Bars represent 100 µm. (F) Number of neutrophils classified according to their speed: “stationary” for neutrophils moving in an area of <10 µm diameter; “rolling” when neutrophils circulate at ≤200 µm/s over 10 µm distance or more; and “circulating” in other cases. Bars represent mean ± SEM. Two-way ANOVA with repeated measures followed by a post-hoc Holm-Bonferroni test (D). *P < .05; **P < .01.

Platelet and neutrophil interactions observed in response to immune complexes. (A-C) Schematic representations (left panels) of different cellular interactions recorded in the brain of both FcγRIIATGN and FcγRIIAnull (Ly6gCre+/−::Rosa26-TdT+/−::CD41-YFP+/−) mice injected with immune complexes and observed with a high-speed widefield fluorescence microscope (right panels). (A) Platelet (cyan) and neutrophil (red) interactions can be associated with partial adhesion (rolling) of neutrophils (neutrophil 2) on blood vessels. (B) “Kiss and fly interaction,” in which an adhered neutrophil (red) briefly interacts with circulating platelets (cyan). (C) Neutrophils (red) adhere to blood vessels in response to immune complexes. Circulating platelets (cyan) interact (white arrows) with both immobilized and rolling neutrophils (yellow arrows). Bars represent 50 µm. The direction of blood flow is indicated with double white arrow heads. Time following the injection of immune complexes is indicated in the inferior right corner (00:00 = minutes:seconds or 00:00:00 = hours:minutes:seconds). Rolling and adhered neutrophils are indicated with yellow arrows, and neutrophil–platelet interactions are indicated with white arrows. (D) Schematic representation of the computer analysis used to track neutrophil trajectories and speed: (1) Image stabilization using Mathworks Matlab 2018b; (2) Neutrophil tracking performed with Bitplane Imaris 7.6 software; (3-4) Automated classification of trajectories using a program in Mathworks Matlab 2018b. (E) Recorded neutrophil trajectories (white lines) in the blood vessels of FcγRIIAnull and FcγRIIATGN mice injected with diluent or immune complexes (Bitplane Imaris 7.6 software). Neutrophils are color coded according to their speed: from dark blue (0 µm/sec) to dark red (4000 µm/sec). Bars represent 100 µm. (F) Number of neutrophils classified according to their speed: “stationary” for neutrophils moving in an area of <10 µm diameter; “rolling” when neutrophils circulate at ≤200 µm/s over 10 µm distance or more; and “circulating” in other cases. Bars represent mean ± SEM. Two-way ANOVA with repeated measures followed by a post-hoc Holm-Bonferroni test (D). *P < .05; **P < .01.

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