Elevated expression of CXCR4 on BM neutrophils from AnxA1−/− mice.
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C) Chemotatic response to various concentrations of CXCL12 was assessed for purified BM neutrophil populations from both WT and AnxA1−/− animals.
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Chemotaxis assay: Directed cell migration potential/chemotaxis towards SDF-1 gradient was analyzed in vitro as described [.
- Briefly, 0.5x106 cells were induced by addition of 1μg/ml doxycyline in IMDM growth media and incubated for 48 hours.
- 600 μl IMDM (supplemented with 10% FBS) containing 125 ng/ml SDF-1α was added to the lower chamber of a Costar 24-wells transwell (pore size 5 μm, ).
- Then, 0.1x106 doxycycline induced cells in 100 μl medium'>IMDM medium (supplemented with 0.1% FBS) were loaded to the upper chamber and allowed to migrate for 4 hours at 37°C in a humidified CO2 incubator.
- After incubation, migrated cells were collected from lower chamber and counted on a hemocytometer under microscope.
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