ATP- and Bz-ATP-induced intracellular calcium fluxes in undifferentiated and neural-differentiated ESC.
ATP- and Bz-ATP-evoked P2X7R responses were determined by single-cell calcium imaging and microfluorimetry assays as described in Materials and Methods. (A) For calcium imaging, 10 µM of purinergic receptor agonists, ATP or Bz-ATP, were used to stimulate receptor-mediated intracellular calcium fluxes in undifferentiated and neural-differentiated cells (day 8). (B) For microfluorimetry-based calcium measurements, increasing concentrations of ATP or Bz-ATP were added and time kinetics of changes in fluorescence emission was recorded. Peak values of measured [Ca2+]i transients are reported. Nonlinear Regression was used for curve-fitting and calculating the EC50 values. (C and D) P2X7R inhibitors were studied regarding their effects in blocking ATP- and Bz-ATP-induced [Ca2+]i transients, as studied by calcium imaging. For these experiments, cells were pre-treated for 2 min with 10 µM KN-62 or 1 µM A438079 and then stimulated with 10 µM ATP or Bz-ATP. Data represent mean values ±S.E. of three independent experiments performed in triplicate.