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Isolation and chemotaxis assay of neutrophils

YC Yuan Cheng
FM Fei Mo
QL Qingfang Li
XH Xuejiao Han
HS Houhui Shi
SC Siyuan Chen
YW Yuquan Wei
XW Xiawei Wei
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All bone marrow cells were harvested from femur and tibia by flushing bone cavities using culture medium without FBS and filtered through 70 μm nylon mesh following RBC lysis. The cell suspension was carefully layered onto Histopaque-1077/1119 gradient solutions (Sigma) and was centrifuged at 700 g for 30 min. After centrifugation, the neutrophils were aspirated from the responding layer in accordance with the manufacturer’s instruction. The cells were then cultured in RPMI 1640 medium supplemented with 10% FBS, penicillin, and streptomycin. Supernatants of LL2 tumor cells were collected 24 h after the cells reached 70–80% density. Isolated neutrophils were seeded in upper chamber of Transwell system, and 1640 RPMI medium, tumor supernatant (TS), CXCL2 or SB225002 were added to the lower chamber. After 6 h of incubation, the number of migrated neutrophils was calculated by flow cytometry.

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