Chemotaxis assay and video imaging

Cells were starved for 5 to 6 h in Soerensen phosphate buffer (23°C, 160 rpm) at a density of 1 x 10⁷ cells/ml. 25–30 μl of the cell suspension was diluted in 3 ml of Soerensen phosphate buffer and mixed well by pipetting (25–30 times, with occasional vortexing). This is important in order to dissociate aggregates. 1.5 ml of the diluted cells were then transferred onto a glass cover-slip with a plastic ring placed on a Leica inverse microscope equipped with a 20x UplanFl 0.3 objective. Cells were stimulated with a glass capillary micropipette (Eppendorf Femtotip) filled with 0.1 mM cAMP [17], which was attached to a microcontroller. Time-lapse images were captured at 30 seconds intervals with a JAI CV-M10 CCD camera and an Imagenation PX610 frame grabber (Imagenation Corp., Beaverton, OR) controlled through Optimas software (Optimas Corp., Bothell, Washington) and stored on a computer hard drive.