Simultaneous measurement of collagen-induced platelet NO production and calcium elevation. X-Rhod1-AM and DAF-FM double-labeled platelets, prepared as described under Methods, were perfused for 3 minutes at 3000 s⁻¹ on a collagen-coated surface and the 2 fluorescence intensities in surface-interacting platelets were monitored for 60 seconds. Representative curves of the fluorescence intensities of single platelets labeled with X-Rhod1-AM and DAF-FM are reported. The analysis of 100 platelets revealed a mean lag phase between the calcium peak and NO signals of 33 ± 9.5 seconds (mean ± 95% CIs).