Murine neutrophils were isolated from 8–12 week old C56BL/6 mice. Mice were euthanized by cervical dislocation, and bone marrow cells were harvested from femur and tibia within 30 min. Cell suspensions were passed through 70 µm cell strainer. Neutrophils were prepared using a negative selection kit (EasySep Mouse Neutrophil Enrichment Kit, Stem Cell Technologies), following manufacturer instruction. Cells were cultured in RPMI 1640 media supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 mM HEPES, and 2 mM EDTA. To stimulate neutrophils, 100 nM phorbol 12-myristate 13-acetate (PMA) (Cayman Chemical) was added to the media.
Human neutrophils were isolated from 8 ml of blood collected from healthy donors. Neutrophils were purified using the MACSxpress Whole Blood Neutrophil Isolation Kit (Miltenyi Biotec 130–104-434) followed by erythrocyte depletion (Miltenyi Biotec 130–098-196) according to manufacturer’s instructions.
Human neutrophils were isolated from 8 ml of blood collected from healthy donors. Neutrophils were purified using the MACSxpress Whole Blood Neutrophil Isolation Kit (Miltenyi Biotec 130–104-434) followed by erythrocyte depletion (Miltenyi Biotec 130–098-196) according to manufacturer’s instructions.