Mouse anti myeloperoxidase

Neutrophils were seeded (at 2x10 5 cells/500µL) in RPMI media plus 2% AB serum in a 24-well plate containing poly-L-lysine coated coverslips as previously described (ChaPMAn et al., 2019) (link). Cells were allowed to adhere for 1h prior to stimulation with phorbol 12-myristate 13-acetate (PMA, 50nM, Sigma), A23187 (3.8µM, Sigma) or 10% RA SF. Cells were incubated for a further 4h to allow for NET production. Cells adhered to coverslips were fixed with 4% paraformaldehyde prior to immunofluorescent staining. Briefly, coverslips were removed from the plate and washed with PBS, permeabilised with 0.05% Tween 20 in TBS, fixed with TBS (2% BSA) and then stained for 30 min on drops of TBS (2% BSA) on parafilm stretched across a clean 24-well plate. Primary antibodies used were mouse anti-myeloperoxidase (1:1000, Abcam) and rabbit anti-citrullinated histone H3 (1:250, Abcam). Coverslips were washed three times with TBS prior to secondary antibody staining (anti-rabbit AlexaFluor488, 1:2000, anti-mouse AlexaFluor647, Life Technologies) in TBS (+2% BSA) for 30 min. Coverslips were washed prior to staining with DAPI (1g/mL, Sigma (Sigma). Slides were imaged on an Epifluorescent microscope (Zeiss) using a 40X objective. Images were analysed using ImageJ (Schindelin et al., 2015) (link) and are presented with equal colour balance.