Goat anti mouse alexa fluor 594

C. jejuni NET induction described above occurred on poly-l-lysine coated glass coverslips (Corning, Cat. 354085) for three hours at 37°C under microaerobic conditions (Volker Brinkmann et al., 2010 (link)). Following three 1x PBS washes, coverslips were incubated with a goat anti-myeloperoxidase (R&D, Cat. AF3174) and donkey anti-goat Alexa Fluor 405 (AbCam, Cat. Ab175664). Coverslips were also incubated with a mouse anti-neutrophil elastase (R&D, Cat. MAB91671) and goat anti-mouse Alexa Fluor 594 (Biolegend, Cat. 405326). After three 1x PBS washes, coverslips were incubated with 1.0 μM SYTOX Green (Invitrogen, Cat. S7020). Finally, after three 1x PBS washes, coverslips were placed on glass slides with a drop of Mowiol mounting medium (Sigma, Cat. 81381–50G) and kept in the dark at 4 °C until fluorescent microscopy was performed. Ten fields of each sample condition were visualized using a Nikon E600 Eclipse at the Advanced Microscopy and Imaging Center at the University of Tennessee. For statistical analysis, three individual neutrophil isolations were performed and stained as described above. Of those, 100 fields were observed for the presence of fluorescent NETs. Statistical analysis was performed using unpaired t tests and significance inferred at p < 0.05.