*2.8. Immunofluorescence Staining*

Isolated BM neutrophils were plated to adhere in fibronectin-coated 96-well plates (Corning) or Nunc glass base dishes (Thermo Fisher Scientific, MA, USA) and pretreated with vehicle, AM281 (10 μM), NAC (5 mM), PTX (5 ng/mL), YM254890 (10 μM) or SB203580 (10 μM) for 20 min, respectively, and stimulated for 2 h with ACEA or JWH133. Neutrophils were fixed by 4% paraformaldehyde for 30 min, after blocked with 2% BSA (Roche, Switzerland), they were incubated with the specific primary antibodies for CitH3 (1:200), MPO (1:200) or Ly-6G (Clone 1A8, 551459, 1:100, BD pharmingen), CB1 (10006590, 1:200, Cayman Chemical, Ann Arbor, MI, USA), CB2 (1:200, 101550, Cayman Chemical) or Rabbit IgG Isotype Control (1:100, 10500C, Invitrogen, CA, USA). Cy3-conjugate affinipure goat-anti-rabbit IgG (111165003, 1:100) or Cy5-conjugate affinipure goat-anti-rat IgG (112175143, 1:100, Jackson Immunoresearch, PA, USA) were as secondary antibodies. For F-actin, neutrophils were fixed by 4% paraformaldehyde for 30 min, penetrated by 0.5% Triton X-100 for 15 min and after blocked with 2% BSA, FITC-conjugated phalloidin (A12379, 1:100, Molecular Probes, OR, USA) was incubated for 20 min. Nuclei were stained with DAPI and SYTOX Green. The sample was observed under confocal microscope (LSM510, Carl Zeiss MicroImaging GmbH, Germany). For high content analysis, the plates were imaged on Thermo Scientific CellInsight personal cell imaging platform (Thermo Fisher Scientific). Fluorescence intensity of each well was analyzed by Cellomics Cell Health Profiling BioApplication software.
