Quantifying Neutrophil Extracellular Traps

ELF-PEMF-exposed neutrophils suspension (150 μL, 3 × 10⁵ cells/mL) were seeded in sterile poly-L-lysine-coated chamber slides. After 4 h of incubation, neutrophils were fixed with 4% formaldehyde for 30 min and incubated with 1% Triton-X-100 for 5 min at room temperature. Fixed neutrophils were gently washed 3 times with PBS, incubated with 5% BSA solution for 1 h, and then incubated with the primary antibody overnight at 4 °C. Anti-MPO antibody (1:200 v/v, Cat. #sc-52707, Santa Cruz Biotechnology, Heidelberg, Germany) was used as the primary antibody. After the overnight incubation, neutrophils were gently washed 3 times with PBS and incubated with Alexa Fluor 488 goat anti-mouse IgG (1:1000 v/v, Cat. #A10667, Invitrogen, Heidelberg, Germany) for 2 h at room temperature. After gently washing 3 times, neutrophils were incubated with the 2 µg/mL Hoechst 33342 for 20 min to visualize the DNA. A fluorescence microscope was used to capture the images (EVOS FL, Life Technologies, Darmstadt, Germany). The ratio of NETosed cells was obtained by dividing the number of MPO⁺ cells by the number of Hoechst 33342⁺ cells. The analysis was performed as previously described [69 (link)].