Cell Invasion Assay with Nicotine

The cell invasion assay was carried out according to our previous study [66 (link)] using 10-well chemotaxis chambers (Neuro Probe, Gaithersburg, Maryland, USA) with an 8-µM pore membrane (Neuro Probe) in RPMI-1640 with 10% FBS as the chemoattractant in the lower chamber. Briefly, AGS cells were added to the upper chamber with nicotine for 24 h, the non-invading cells on the upper surface of each membrane were removed from the chamber by using cotton swabs, and the invading cells on the lower surface of each membrane were stained using the Quick-Diff stain kit (Becton-Dickinson, Franklin Lakes, NJ, USA). After two washes with water, the chambers were allowed to air dry. The number of invading cells was counted using a phase-contrast microscope.