Poly lysine coated 24 well glass bottom plates

Another method commonly used to confirm the production of NETs is staining the samples with antibodies against myeloperoxidase (MPO). MPO is an antimicrobial protein found on the chromatin fibers released during NETosis.
Isolated human neutrophils (5.5 × 10⁵ cells/ml) were suspended in RPMI and added to poly‐lysine‐coated 24 well glass bottom plates (Cellvis, CA) and incubated at 37°C in 5% CO₂ for 10 min. After stimulation with PMA (50 nM) or LPS from P. gingivalis or E. coli (1 μg/ml), the cells were further incubated for 3 hr (the incubation time was chosen from the results received after Sytox Green DNA stain). Cells were fixed in 4% paraformaldehyde for 30 min at room temperature and permeabilized with cold acetone and methanol (1:1) for 5 min. To visualize NETs, the samples were stained with antibodies against MPO (DAKO), followed by secondary antibody staining (Donkey Anti‐Rabbit IgG H&L Alexa Fluor 488 purchased from ThermoFisher). Finally, the coverslips were mounted with ProLong Gold antifade mountant with DAPI (ThermoFisher). The cells were visualized using an Olympus BX41 epifluorescent microscope with the CellSens software.

Isolated human neutrophils (5.5 × 10⁵ cells/ml) were suspended in RPMI and added to poly‐lysine‐coated 24 well glass bottom plates (Cellvis, California, USA) and incubated at 37°C in the presence of 5% CO₂ for 10 min. After stimulation with PMA (50 nM) or the methacrylates, the cells were further incubated at 37°C in the presence of 5% CO₂ for 3 hr (the incubation time was chosen from the results received after Sytox Green DNA stain). Cells were fixed in 4% paraformaldehyde for 30 min at room temperature and, permeabilized with cold acetone and methanol (1:1) for 5 min. To visualize NETs, the samples were stained with antibodies against myeloperoxiadase (MPO, DAKO), followed by secondary antibody staining (Donkey Anti‐Rabbit IgG H&L Alexa Fluor® 488 purchased from ThermoFisher). Finally, the coverslips were mounted with ProLong™ Gold antifade mountant with the DNA stain DAPI (ThermoFisher). The cells were visualized using an Olympus BX41 epifluorescent microscope with the CellSens software.