Immunofluorescence Staining of NETs

Identification of NETs in this study was similar to a previously published protocol.^{10 (link)} Briefly, 2 × 10⁵ BMDNs were seeded onto poly-l-lysine-coated coverslips (Sigma-Aldrich). In experiments without stimulation or with stimulation with 2% serum (extracted from pristane-treated WT or Mfge8^−/− mice for 6 months), the cells were incubated for 2 h. In experiments with 1 μg/ml LPS, 100 nM PMA or 100 ng/ml MIP-2 stimulation, the incubation was for 4 h. NETs were stained with rabbit anti-histone H3 (citrulline R2+R8+R17) antibody and mouse anti-MPO antibody, followed by incubation with Alexa Fluor 647-conjugated mouse IgG and Alexa Fluor 488-conjugated rabbit IgG (Abcam). DNA was stained with DAPI. Images were collected with Olympus BX51 microscope and Qimaging camera, typically at original × 400 magnification.