TC-derived soluble factors modified neutrophils’ kinetic properties.

Neutrophils were stimulated with a TC-CM or the control medium for 18 hours. Within this time window, digital phase contrast images of 15 fields/well were captured every 15 minutes via a 20× objective in the Operetta high-content imaging system. PhenoLOGIC (PerkinElmer) was employed for image segmentation and to calculate the single-cell kinetic properties (A), accumulated distance, (B) speed and straightness (C) in dedicated analysis sequence. The results were expressed as mean ± SEM of six independent experiments; ***p < 0.005; **p < 0.01; *p < 0.05. D-E. Timepoint analyses of time-dependent properties such as current step size (D) and current speed (E) illustrating dynamic changes of the behavior of neutrophils (in presence or absence of TC-CMs) in function of the elapsed time from treatment. The results were expressed as mean ± SEM of six independent experiments; ****p < 0.001.