Figure 5.

Neutrophils isolated from mouse pups fail to form NETs; a recapitulation of human peripartum NETosis inhibition. (A) NET formation in response to LPS stimulation (100 ng/mL; 1 hour) was detected in mouse PMNs isolated from C57BL/6 pups on days 0, 5, 8, 10, and 16 following birth. Confocal microscopy images of NET formation were obtained by using DNA stains, one that is cell permeable (SYTO Green, nuclear DNA, green) and the other that is cell impermeable (SYTOX Orange, NETs, magenta). Images are representative of 4 to 7 different experiments in PMNs isolated from 4 to 7 separate mouse pups per group. Scale bar, 10 μm. (B) A grid system was used to semiquantitatively assess NET formation. The y-axis depicts NET formation with NETs crossing standardized grid lines/high-power field (hpf) (± SEM). (C) Plasma “switch” experiments were performed, and NET formation was assessed by using PMNs isolated from mature mice ± a 1-hour pretreatment with day 0 mouse pup plasma. PMNs were then stimulated with LPS (100 ng/mL; 1 hour) and live cell imaging performed as in panel A. Images are representative of 3 different experiments in PMNs isolated from 3 separate mouse pups per group. Yellow arrows indicate NET formation. Scale bars, 50 μm (10 μm for the 20× and 60× magnification images, respectively). (D) A grid system was used to semiquantitatively assess NET formation. The y-axis depicts NET formation with NETs crossing standardized grid lines/hpf (± SEM) . *P < .05 and ****P < .0001 for comparison of day 8 and days 10 and 16 with day 0 and day 5, respectively; ***P < .001.