Bradyzoite-Induced NETosis and LC3 Expression

LC3 has been used as a classical marker for autophagosomes (Karim et al., 2007 (link)), being LC3-I cytosolic and LC3-II membrane bound and enriched in the autophagic vacuole. Therefore, we tested whether LC3 expression might be present during B. besnoiti bradyzoite-induced NETosis as described elsewhere (Zhou et al., 2019 (link)). Briefly, bovine PMN (n = 3) were added on 0.01% poly-_L-lysine pre-coated coverslips (15 mm diameter; Nunc), then stimulated by B. besnoiti bradyzoites for 1 h at RT. After incubation, cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with cold methanol (Merck) for 3 min and blocked by using the following blocking buffer (5% BSA (Sigma-Aldtich), 0.1% Triton X-100 (Sigma-Aldrich) in sterile PBS) for 60 min at RT. After removing blocking buffer, cells were incubated overnight at 4°C with rabbit anti-LC3B antibodies (Cat#2775, 1:200, Cell Signaling Technology) diluted in blocking buffer, washed three times with PBS, incubated with goat anti-rabbit IgG conjugated with Alexa Fluor 488 (Invitrogen) for 1 h in the dark at RT. After being washed three times with PBS, coverslips were mounted by prolonged anti-fading reagent with DAPI (Invitrogen) on glass slides (Nunc), and five randomly images were taken per condition using an inverted epifluorescence microscope IX 81^® (Olympus) and/or by using confocal microscopy analysis (LSM 710^®; Zeiss).