Inducing Chemotactic Competence in Dictyostelium

Cells expressing PHcrac-GFP were made chemotactically competent as described [48 (link)]. Briefly, cells were washed twice in development buffer (DB) before being resuspended in DB to 2 × 10⁷ cells/mL and rotated for 1 h at 110 rpm. Cells were pulsed with 60 nM of cAMP (Sigma, Gillingham, Dorset, UK, A6885) every 6 min for 4 h, in the presence or absence of the compound (60 µM decanoic acid or octanoic acid). Cells were basalated by the addition of caffeine (Sigma, Gillingham, Dorset, UK, C0750) to 5 mM, with rotation at 200 rpm for 30 min, before being washed twice in cold DB buffer and resuspended to 2 × 10⁷ cells/mL. For time-lapse imaging of PIP₃ production, 360 µL chemotactically competent cells expressing PHcrac-GFP at 5 × 10⁵ cells/mL were placed into a well of an eight-well chambered cover glass (PI3K inhibitor LY294002 (Selleckchem, Houston, TX, USA, S1105) was added to one sample at a final concentration of 100 µM) and left to adhere for 10 min. Cells were stimulated by adding cAMP, to a final concentration of 1 µM, and images were captured every 2 s for 1 min.