RvTs limit S aureus infection and reduce NETs in vivo. Mice were given a panel of RvTs (RvT1, RvT2, RvT3, and RvT4; 50 ng each) or vehicle control together with live S aureus (10⁷ CFU) by intra-pouch injection. Sixteen hours later, pouch exudates were collected. (A) Exudate leukocytes were enumerated by using light microscopy and PMN percentages determined by differential counting. Mean ± SEM. n = 7 (leukocytes) or n = 4 (PMNs). *P < .05, **P < .01, two-tailed Student t test. (B) Exudate bacterial titers were determined by enumerating colonies on LB agar plates. Mean ± SEM. n = 6. *P < .05, two-tailed Student t test. (C) NETs were quantified by using the microfluidic NET devices. Exudate cells (2 × 10⁵ cells) were incubated with Sytox Green (5 µM) for 15 minutes and loaded onto the microfluidic NET device. NETs were quantified according to 2 criteria: (left) long-string NETs > 100 μm² (fluorescent areas larger than 100 μm² in size with a shape circularity 0-0.5) or (right) NETs > 100 μm² (fluorescent areas larger than 100 μm² in size with shape circularity 0-1). Mean ± SEM. n = 5 or 6. *P < .05, two-tailed Student t test. (D) Representative images of NETs > 100 μm² and shape circularity 0-1. (E) Representative images. Exudate cells (2 × 10⁵ cells) were adhered onto 8-well chamber slides, incubated with Sytox Green (5 µM), followed by staining with (left panels) phycoerythrin-conjugated anti-Ly6G antibody for mouse PMN or (right panels) goat anti-mouse MPO antibody. Arrows denote NETs. Scale bars = 50 μm. (F) NETs were quantified by using a fluorescence plate reader. Exudate cells (1 × 10⁵ cells) were adhered onto a 96-well plate, incubated with Sytox Green (5 µM) for 20 minutes, and fluorescence determined. Mean ± SEM. n = 7. *P < .05, two-tailed Student t test. Veh, vehicle control.