FITC-collagen is protected from trypan blue quenching in live, TRAP-stimulated platelets. Human platelets were incubated with 25 μg/mL FITC-collagen in the presence or absence of 25 μM TRAP at room temperature for 30 minutes as described in “Materials and methods.” In another group, platelets were fixed with PFA after incubation with FITC-collagen to stop the reaction and to prevent trypan blue exclusion. Platelets were then washed and diluted 1:1 with ice-cold PBS. To compare the fluorescence with or without trypan blue quenching, cell suspensions were mixed 1:1 with trypan blue (400 μg/mL in PBS) or PBS for 1 to 5 minutes before flow cytometry. Data shown are averaged normalized mean fluorescence ± SEM from 2 to 4 experiments. *P < .05, **P > .05, compared with PBS controls.