Prolonggold 4 6 diamidino 2 phenylindole dapi

Neutrophil extracellular trap visualization was performed as described previously (29 (link)). Briefly, 5 × 10⁶ neutrophils were infected with GAS WT and ΔSLO mutant was added at MOI = 1 and incubated for 4 h in 37°C/5% CO₂. Cells were fixed with 4% paraformaldehyde and stained with anti-myeloperoxidase (MPO) antibody (1:300 diluted, Calbiochem) in PBS + 2% bovine serum albumin (BSA, Sigma) for 1 h at RT, followed by incubation with goat anti-rabbit Alexa 488 antibody (1:500 dilution, Life Technologies). Cells were counterstained with ProlongGold + 4′,6′-diamidino-2-phenylindole (DAPI, Invitrogen) and imaged on a fluorescent microscope. Representative, randomized images (n = 5) were taken for each condition and individual experiment. The ratio of NET-releasing cells to non-NET-releasing cells was determined as % of total cells releasing NETs.

Coverslips with adherent monolayer cells were rinsed twice in PBS, fixed with ice-cold methanol for 5 min at −20 °C and then washed again. The coverslips were blocked with 3% Bovine serum albumin (BSA) for 30 min at room temperature, and then incubated with the anti-OPCML (mouse mAb, #MAB27771, R&D Systems) antibody diluted 1:100 at room temperature for 1 h in a wet chamber, washed and incubated with a suitable secondary antibody diluted 1:400 at room temperature for 30 min. Coverslips were mounted with a drop of Prolong gold 4′, 6-diamidino-2-phenylindole (DAPI) (Invitrogen) and left to dry in the dark at room temperature overnight before being examined under an SP5 confocal microscope (Leica).