Neutrophil Activation and Imaging

Neutrophils were incubated with either propofol or lipid emulsion for 30 min at 37°C with 5% CO₂ prior to addition of 25 nM phorbol 12-myristate 13-acetate (PMA; Sigma). Following an additional 2-h incubation at 37°C with 5% CO₂, cells were fixed via addition of paraformaldehyde (4% final concentration). Fixed neutrophils were blocked with PBS plus 2% bovine serum albumin and goat serum, followed by staining using an anti-human myeloperoxidase antibody (1:300; Dako Denmark) and an Alexa 488-labeled secondary antibody (1:500; Life Technologies) as previously described (Corriden et al., 2015 (link)). Cells were imaged using an AxioObserver D1 Inverted Fluorescence Microscope (Zeiss) equipped with an LD A-Plan 20X/0.35 Ph1 objective and processed with affinity photo (Serif).